A Cysteine-Rich Protein, SpDIR1L, Implicated in S-RNase-Independent Pollen Rejection in the Tomato (Solanum Section Lycopersicon) Clade

Tomato clade species (Solanum sect. Lycopersicon) display multiple interspecific reproductive barriers (IRBs). Some IRBs conform to the SI x SC rule, which describes unilateral incompatibility (UI) where pollen from SC species is rejected on SI species’ pistils, but reciprocal pollinations are successful. However, SC x SC UI also exists, offering opportunities to identify factors that contribute to S-RNase-independent IRBs. For instance, SC Solanum pennellii LA0716 pistils only permit SC Solanum lycopersicum pollen tubes to penetrate to the top third of the pistil, while S. pennellii pollen penetrates to S. lycopersicum ovaries. We identified candidate S. pennellii LA0716 pistil barrier genes based on expression profiles and published results. CRISPR/Cas9 mutants were created in eight candidate genes, and mutants were assessed for changes in S. lycopersicum pollen tube growth. Mutants in a gene designated Defective in Induced Resistance 1-like (SpDIR1L), which encodes a small cysteine-rich protein, permitted S. lycopersicum pollen tubes to grow to the bottom third of the style. We show that SpDIR1L protein accumulation correlates with IRB strength and that species with weak or no IRBs toward S. lycopersicum pollen share a 150 bp deletion in the upstream region of SpDIR1L. These results suggest that SpDIR1L contributes to an S-RNase-independent IRB.

Neofunctionalisation of the Sli gene leads to self-compatibility and facilitates precision breeding in potato

Genetic gain in potato is hampered by the heterozygous tetraploid genome of cultivated potato. Converting potato into a diploid inbred-line based F1-hybrid crop provides a promising route towards increased genetic gain. The introduction of a dominant S-locus inhibitor (Sli) gene into diploid potato germplasm allows efficient generation of self-fertilized seeds and thus the development of potato inbred lines. Little is known about the structure and function of the Sli locus. Here we describe the mapping of Sli to a 12.6 kb interval on chromosome 12 using a recombinant screen approach. One of two candidate genes present in this interval shows a unique sequence that is exclusively present in self-compatible lines. We describe an expression vector that converts self-incompatible genotypes into self-compatible and a CRISPR-Cas9 vector that converts SC genotypes into SI. The Sli gene encodes an F-box protein that is specifically expressed in pollen from self-compatible plants. A 533 bp insertion in the promotor of that gene leads to a gain of function mutation, which overcomes self-pollen rejection.

Pollen-Pistil Interactions as Reproductive Barriers

Pollen-pistil interactions serve as important prezygotic reproductive barriers that play a critical role in mate selection in plants. Here, we highlight recent progress toward understanding the molecular basis of pollen-pistil interactions as reproductive isolating barriers. These barriers can be active systems of pollen rejection, or they can result from a mismatch of required male and female factors. In some cases, the barriers are mechanistically linked to self-incompatibility systems, while others represent completely independent processes. Pollen-pistil reproductive barriers can act as soon as pollen is deposited on a stigma, where penetration of heterospecific pollen tubes is blocked by the stigma papillae. As pollen tubes extend, the female transmitting tissue can selectively limit growth by producing cell wall–modifying enzymes and cytotoxins that interact with the growing pollen tube. At ovules, differential pollen tube attraction and inhibition of sperm cell release can act as barriers to heterospecific pollen tubes.

Ornithine decarboxylase genes contribute to S-RNase-independent pollen rejection

Unilateral incompatibility (UI) manifests as pollen rejection in the pistil, typically when self-incompatible (SI) species are pollinated by self-compatible (SC) relatives. In the Solanaceae, UI occurs when pollen lack resistance to stylar S-RNases, but other, S-RNase-independent mechanisms exist. Pistils of the wild tomato Solanum pennellii LA0716 (SC) lack S-RNase yet reject cultivated tomato (S. lycopersicum, SC) pollen. In this cross, UI results from low pollen expression of a farnesyl pyrophosphate synthase gene (FPS2) in S. lycopersicum. Using pollen from fps2-/- loss-of-function mutants in S. pennellii, we identified a pistil factor locus, ui3.1, required for FPS2-based pollen rejection. We mapped ui3.1 to an interval containing 108 genes situated on the IL 3-3 introgression. This region includes a cluster of ornithine decarboxylase (ODC2) genes, with four copies in S. pennellii, versus one in S. lycopersicum. Expression of ODC2 transcript was 1034-fold higher in S. pennellii than in S. lycopersicum styles. Pistils of odc2-/-knockout mutants in IL 3-3 or S. pennellii fail to reject fps2 pollen and abolish transmission ratio distortion (TRD) associated with FPS2. Pollen of S. lycopersicum express low levels of FPS2 and are compatible on IL 3-3 pistils, but incompatible on IL 12-3 x IL 3-3 hybrids, which express both ODC2 and ui12.1, a locus thought to encode the SI proteins HT-A and HT-B. TRD observed in F2 IL 12-3 x IL 3-3 points to additional ODC2-interacting pollen factors on both chromosomes. Thus, ODC2 genes contribute to S-RNase independent UI and interact genetically with ui12.1 to strengthen pollen rejection.

Mechanism of self/nonself-discrimination in Brassica self-incompatibility

Self-incompatibility (SI) is a breeding system that promotes cross-fertilization. In Brassica, pollen rejection is induced by a haplotype-specific interaction between pistil determinant SRK (S receptor kinase) and pollen determinant SP11 (S-locus Protein 11, also named SCR) from the S-locus. Although the structure of the B. rapa S9-SRK ectodomain (eSRK) and S9-SP11 complex has been determined, it remains unclear how SRK discriminates self- and nonself-SP11. Here, we uncover the detailed mechanism of self/nonself-discrimination in Brassica SI by determining the S8-eSRK–S8-SP11 crystal structure and performing molecular dynamics (MD) simulations. Comprehensive binding analysis of eSRK and SP11 structures reveals that the binding free energies are most stable for cognate eSRK–SP11 combinations. Residue-based contribution analysis suggests that the modes of eSRK–SP11 interactions differ between intra- and inter-subgroup (a group of phylogenetically neighboring haplotypes) combinations. Our data establish a model of self/nonself-discrimination in Brassica SI.

Autophagy is required for self-incompatible pollen rejection in two transgenic Arabidopsis thaliana accessions

Successful reproduction in the Brassicaceae is mediated by a complex series of interactions between the pollen and the pistil, and some species have an additional layer of regulation with the self-incompatibility trait. While the initial activation of the self-incompatibility pathway by the pollen S-locus protein11/S-locus cysteine-rich peptide and the stigma S Receptor Kinase is well characterized, the downstream mechanisms causing self-pollen rejection are still not fully understood. In previous studies, we had detected the presence of autophagic bodies with self-incompatible pollinations in Arabidopsis lyrata and transgenic A. thaliana lines, but it was not known if autophagy was essential for self-pollen rejection. Here, we investigated the requirement of autophagy in this response by crossing mutations in the essential AUTOPHAGY7 (ATG7) gene into two different transgenic self-incompatible A. thaliana lines in the Col-0 and C24 accessions. By using these previously characterized transgenic lines that express A. lyrata and A. halleri self-incompatibility genes, we demonstrated that disrupting autophagy can weaken their self-incompatible responses in the stigma. When the atg7 mutation was present, an increased number of self-incompatible pollen were found to hydrate and form pollen tubes that successfully fertilized the self-incompatible pistils. Additionally, we confirmed the presence of GFP-ATG8a labelled autophagosomes in the stigmatic papillae following self-incompatible pollinations. Together, these findings support the requirement of autophagy in the self-incompatibility response and add to the growing understanding of the cellular events that take place in the stigma to reject self-pollen.One Sentence SummaryIn self-incompatible transgenic Arabidopsis thaliana lines, autophagy is an integral part of the cellular responses in the stigma to efficiently block fertilization by self-incompatible pollen.

Intraspecific Genetic Variation Underlying Postmating Reproductive Barriers between Species in the Wild Tomato Clade (Solanum sect. Lycopersicon)

A goal of speciation genetics is to understand how the genetic components underlying interspecific reproductive barriers originate within species. Unilateral incompatibility (UI) is a postmating prezygotic barrier in which pollen rejection in the female reproductive tract (style) occurs in only one direction of an interspecific cross. Natural variation in the strength of UI has been observed among populations within species in the wild tomato clade. In some cases, molecular loci underlying self-incompatibility (SI) are associated with this variation in UI, but the mechanistic connection between these intra- and inter-specific pollen rejection behaviors is poorly understood in most instances. We generated an F2 population between SI and SC genotypes of a single species, Solanum pennellii, to examine the genetic basis of intraspecific variation in UI against other species, and to determine whether loci underlying SI are genetically associated with this variation. We found that F2 individuals vary in the rate at which UI rejection occurs. One large effect QTL detected for this trait co-localized with the SI-determining S-locus. Moreover, individuals that expressed S-RNase—the S-locus protein involved in SI pollen rejection—in their styles had much more rapid UI responses compared with those without S-RNase protein. Our analysis shows that intraspecific variation at mate choice loci—in this case at loci that prevent self-fertilization—can contribute to variation in the expression of interspecific isolation, including postmating prezygotic barriers. Understanding the nature of such intraspecific variation can provide insight into the accumulation of these barriers between diverging lineages.

Generation of Transgenic Self-Incompatible Arabidopsis thaliana Shows a Genus-Specific Preference for Self-Incompatibility Genes

Brassicaceae species employ both self-compatibility and self-incompatibility systems to regulate post-pollination events. Arabidopsis halleri is strictly self-incompatible, while the closely related Arabidopsis thaliana has transitioned to self-compatibility with the loss of functional S-locus genes during evolution. The downstream signaling protein, ARC1, is also required for the self-incompatibility response in some Arabidopsis and Brassica species, and its gene is deleted in the A. thaliana genome. In this study, we attempted to reconstitute the SCR-SRK-ARC1 signaling pathway to restore self-incompatibility in A. thaliana using genes from A. halleri and B. napus, respectively. Several of the transgenic A. thaliana lines expressing the A. halleri SCR13-SRK13-ARC1 transgenes displayed self-incompatibility, while all the transgenic A. thaliana lines expressing the B. napus SCR1-SRK1-ARC1 transgenes failed to show any self-pollen rejection. Furthermore, our results showed that the intensity of the self-incompatibility response in transgenic A. thaliana plants was not associated with the expression levels of the transgenes. Thus, this suggests that there are differences between the Arabidopsis and Brassica self-incompatibility signaling pathways, which perhaps points to the existence of other factors downstream of B. napus SRK that are absent in Arabidopsis species.