Robust Self-Incompatibility in the Absence of a Functional ARC1 Gene in Arabidopsis thaliana

Abstract

Self-incompatibility (SI) is the primary determinant of the outbreeding mode of sexual reproduction in the Brassicaceae. All Arabidopsis thaliana accessions analyzed to date carry mutations that disrupt SI functions by inactivating the SI specificitydetermining S locus or SI modifier loci. S-locus genes isolated from self-incompatible close relatives of A. thaliana restore robust SI in several accessions that harbor only S-locus mutations and confer transient SI in accessions that additionally harbor mutations at modifier loci. Self-incompatible transgenic A. thaliana plants have proved to be valuable for analysis of the recognition and signaling events that underlie SI in the Brassicaceae. Here, we review results demonstrating that S-locus genes are necessary and sufficient for SI signaling and for restoration of a strong and developmentally stable SI phenotype in several accessions of A. thaliana. The data indicate that introduction of a functional E3 ligase-encoding ARC1 gene, which is deleted in all accessions that have been analyzed to date, is not required for SI signaling leading to inhibition of self pollen or for reversion of A. thaliana to its fully self-incompatible ancestral state.It is well established that specific pollen recognition in the self-incompatibility (SI) response of the Brassicaceae is determined by allele-specific interactions that occur at the stigma surface between two highly polymorphic proteins encoded in the S locus: the S-locus receptor kinase SRK and its ligand, the S-locus cysteine-rich protein SCR. Arabidopsis thaliana lacks a functional SI system and harbors nonfunctional S-locus variants that contain defective alleles of the SRK and/or SCR genes (Kusaba et al., 2001; ShermanBroyles et al., 2007;Tang et al., 2007;Shimizu et al., 2008;Boggs et al., 2009a;Tsuchimatsu et al., 2010;Dwyer et al., 2013). Despite being highly self-fertile, A. thaliana can be made to express SI upon transformation with functional SRK-SCR gene pairs isolated from its self-incompatible close relatives (Nasrallah et al., 2002(Nasrallah et al., , 2004Boggs et al., 2009aBoggs et al., , 2009b. The first transfer of the SI trait into A. thaliana was achieved using the SRKbSCRb gene pair isolated from the Sb locus of Arabidopsis lyrata (Kusaba et al., 2001;Nasrallah et al., 2002Nasrallah et al., , 2004 (Gu et al., 1998), and it was subsequently inferred to be required for SI because downregulation of the ARC1 gene in B. napus (Stone et al., 1999) and A. lyrata (Indriolo et al., 2012), as well as overexpression of ARC1's target, Exo70A1, in B. napus (Samuel et al., 2009), caused partial breakdown of the SI response. However, the involvement of the proposed SRK-ARC1-Exo70A1 pathway in SI has been questioned because the ARC1 gene was found to be deleted in all A. thaliana accessions analyzed to date (Kitashiba et al., 2011;Indriolo et al., 2012), including those in which the SRKb-SCRb transgenes confer a strong SI phenotype (Kitashiba et al., 2011). Additionally, overexpression of Exo70A1 did not cause weakenin...

The ARC1 E3 Ligase Promotes a Strong and Stable Self-Incompatibility Response in Arabidopsis Species: Response to the Nasrallah and Nasrallah Commentary

“…We agree with Nasrallah and Nasrallah (2014) that the pollination analyses should include aniline blue staining for pollen tubes ( Figure 1B). Given that self-pollen rejection occurs rapidly at the stigmatic surface, we additionally would like to see corresponding differential interference contrast images of these aniline blue-stained pollinated pistils to view the elongated stigmatic papillae for correct staging and the number of adhered pollen grains.…”

“…All of these transformants also displayed a corresponding decrease in attached pollen grains and pollen tube growth on self-pollinated pistils. Nasrallah and Nasrallah (2014) argue that ARC1 is not needed as the reduced amount of seed set that they observed is lower than what we reported. In our qRT-PCR analyses, equivalent SCRb and SRKb expression levels were observed in the self-incompatible Sha:AlSCRb-SRKb transformants versus the Sha:AlSCRb-SRKb1ARC1 transformants .…”

“…In terms of the recommendations for the adoption of a standardized strategy for functional studies of self-incompatibility in A. thaliana, Nasrallah and Nasrallah (2014) recommended the use of "well-defined true breeding SCR-SRK lines." We feel that in presenting new data, multiple independently transformed plants should be studied in both the T1 and T2 generations to ensure that the reported phenotypes are reproducible and segregate with the introduced transgenes in progeny of the primary transformants.…”

“…We demonstrated a conserved role for ARC1 in self-incompatible Brassica napus (Gu et al, 1998;Stone et al, 1999Stone et al, , 2003, self-incompatible Arabidopsis lyrata (Indriolo et al, 2012), and self-incompatible Arabidopsis thaliana expressing A. lyrata SCRb, SRKb, and ARC1 transgenes . The commentary by Nasrallah and Nasrallah (2014) focuses on our proposed role for ARC1 in reconstituting self-incompatibility in transgenic A. thaliana.…”

Flowers and mycorrhizal roots – closer than we think?

“…by S-locus receptor kinase (SRK) which activates the abortion of self-pollen by a mechanism that is currently under intense debate [45][46][47][48]. The extracellular domain of SRK contains a highly conserved cysteine-rich stretch consisting of an EGF-like and a PAN_APPLE motive that are required for receptor dimerization [49], and comprises Nglycosylation sites that are essential for localization to the plasma membrane [50].…”