Investigating a role for PUB17 and PUB16 in the self-incompatibility signaling pathway in transgenic Arabidopsis thaliana

Abstract

In Brassicaceae self-incompatibility (SI), self-pollen rejection is initiated by the S-haplotype specific interactions between the pollen S cysteine-rich/S-locus protein 11 (SCR/SP11) ligands and the stigma S receptor kinases (SRK). In Brassica SI, a member of the Plant U-Box (PUB) E3 ubiquitin ligases, ARM-repeat containing 1 (ARC1), is then activated by SRK in this stigma and cellular events downstream of this cause SI pollen rejection by inhibiting pollen hydration and pollen tube growth. During the transition to selfing, Arabidopsis thaliana lost the SI components, SCR, SRK, and ARC1. However, this trait can be reintroduced into A. thaliana by adding back functional copies of these genes from closely related SI species. Both SCR and SRK are required for this, though the degree of SI pollen rejection varies between A. thaliana accessions, and ARC1 is not always needed to produce a strong SI response. For the A. thaliana C24 accession, only transforming with Arabidopsis lyrata SCR and SRK confers a strong SI trait (SI-C24), and so here, we investigated if ARC1-related PUBs were involved in the SI pathway in the transgenic A. thaliana SI-C24 line. Two close ARC1 homologs, PUB17 and PUB16, were selected, and (Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology was used to generate pub17 and pub16 mutations in the C24 accession. These mutants were then crossed into the transgenic A. thaliana SI-C24 line and their potential impact on SI pollen rejection was investigated. Overall, we did not observe any significant differences in SI responses to implicate PUB17 and PUB16 functioning in the transgenic A. thaliana SI-C24 stigma to reject SI pollen.

Keywords: ARC1; Arabidopsis; Brassica; PUB16; PUB17; SRK; self‐incompatibility.

FIGURE 1

Phylogenetic analysis of predicted U‐box N‐terminal domain (UND)‐plant U‐Box (PUB) proteins in Arabidopsis thaliana and Arabidopsis lyrata . The amino acid sequences for A. thaliana UND‐PUBs are shown in blue and A. lyrata UND‐PUBs are in green (the A. lyrata ARM‐repeat containing 1 [ARC1] name is in red). The yellow box highlights the clustering of A. lyrata ARC1 with PUB17 and PUB16. The maximum likelihood tree was built in Molecular Evolutionary Genetics Analysis Version 11 (MEGA11) using default parameters and 1000 bootstrap replications.

FIGURE 2

Gene expression of PUB17 and PUB16 in Arabidopsis thaliana. (a) RT‐PCR analysis of PUB17 and PUB16 expression in pistil tissues from A. thaliana Col‐0 and C24. cDNA was synthesized from RNA extracted from the top half of pistils (including the stigmas), and RT‐PCR was conducted to confirm the expression of PUB17 and PUB16 in both the Col‐0 and C24 accessions. Elongation factor 1α2 (EF1A2) was used as a positive control. PUB17 = 467 bp, PUB16 = 305 bp, EF1A2 = 355 bp. (b) Schematic representation of CRISPR/Cas9 deletion mutants for PUB17 and PUB16. Both genes are encoded by a single exon (gray rectangle), and dashed lines represent deletions in the mutant alleles. Overlaid on the coding regions are the three predicted protein domains (U‐box N‐terminal domain [UND], U‐box, ARM domain) from Mudgil et al. (2004) for PUB17 and PUB16. Both of the pub17 mutations are predicted to have in‐frame deletions and the predicted partial remaining UND and ARM domains are shown. Both of the pub16 mutations are predicted to have frameshifts with premature stop codons (*) following the deletions and only contain predicted UND domains as shown. Scale bars = 250 bp.

FIGURE 3

Pollen hydration assays on SI‐C24 and SI‐C24 pub mutant stigmas. (a–c) SI‐C24 pollen hydration assays on stigmas from (a) C24, SI‐C24, pub17, and SI‐C24 pub17 mutants; (b) C24, SI‐C24, pub16, and SI‐C24 pub16 mutants; and (c) C24, SI‐C24, and the SI‐C24 pub16‐1 pub17‐5 mutant. (d) C24 pollen hydration assays on stigmas from C24 and the SI‐C24 pub16‐1 pub17‐5 mutant. Pollen diameters were measured at 0‐ and 10‐min post‐pollination. Data are shown as bar graphs of the means ± SE with all the data points displayed. n = 30 pollen grains per line. Letters represent statistically significant groupings of p < .05 based on a one‐way analysis of variance (ANOVA) with a Tukey's honest significance difference (HSD) post‐hoc test. Pollen hydration assays on stigmas from C24 and the individual pub mutants (a, b) and C24 pollen on stigmas from SI‐C24 pub16‐1 pub17‐5 (d) represent control pollinations to confirm the single and double pub mutations did not impact compatible pollen hydration. The single and double pub mutations also did not impact the stigma SI response at the level of pollen hydration (a–c).

FIGURE 4

Aniline blue images of SI‐C24 pollinated SI‐C24 and SI‐C24 pub mutant pistils. (a–p) Representative images of pistils collected at 24‐h post‐pollination for aniline blue staining. The pistils were pollinated with SI‐C24 pollen, except for (m, n) which were pollinated with C24 pollen. The genotypes of the pistils are indicated above, and brightfield (left) and aniline blue images (right) are shown for each sample. Pollen tubes growing through the pistil are indicative of pollen acceptance as shown in panels (b, f, j, n) which represent compatible pollination controls. The pub mutations also had no impact on the stigma SI response as observed by the lack of SI‐C24 pollen tube growth (panels d, h, l, p). Scale bars = 100 μm. (q) Bar graph showing the mean number of pollen tubes/pistil at 24‐h post‐pollination. Data are shown as mean ± SE with all the data points displayed. n = 8–16 pistils per line. Letters represent statistically significant groupings of p < .05 based on a one‐way analysis of variance (ANOVA) with a Tukey's honest significance difference (HSD) post‐hoc test.

FIGURE 5

Seed yield for SI‐C24 and SI‐C24 pub mutant siliques. Pistils were pollinated with C24 pollen (left) or SI‐C24 pollen (right), and siliques were harvested at 2‐weeks post‐pollination for counting. Data are shown as a bar graph of the means ± SE with all data points displayed. n = 10–11 siliques per line. Letters represent statistically significant groupings of p < .05 based on a one‐way analysis of variance (ANOVA) with a Tukey's honest significance difference (HSD) post‐hoc test. The pub mutations do not impact seed set upon pollinations with compatible C24 pollen and self‐incompatible SI‐C24 pollen.