Protein function and interactions in gametophytic self-incompatibility : collaborative recognition of S-RNase in vivo

My dissertation is based on studies of Gametophytic Self-Incompatibility (GSI), a system that allows plants to reject self pollen while accepting non-self pollen, thus preventing inbreeding and promoting genetic diversity in populations. In GSI, pollen grains deposited on the stigma of the floral pistil germinate and begin to grow through the transmitting tract tissue of the style. As the pollen tubes grow through the transmitting tract, they import recognition variants of a secreted protein known as the S-locus ribonuclease (S-RNase). If there is a match of recognition specificity between the pollen tube and the imported S-RNase, the S-RNase will degrade pollen-tube RNA, inhibiting protein synthesis & pollen tube growth. Conversely, if there is no match between pollen tube and S-RNase, the action of the S-RNase is inhibited, and the pollen tube continues to grow normally to the ovary. Inside pollen tubes, non-self S-RNases are recognized by the SCF SLF complex comprising multiple variants of the pollen-recognition protein named SLF, along with three other proteins: SSK1, SBP1 and Cullin-1. I have been using protein-interaction assays (BiFC assays) based on the reconstitution of a fluorescent protein, to study interactions between components of the SCFSLF complex and S-RNase. Previous studies revealed that multiple SLF genes collaborate during non-self S-RNase recognition. Based on my data, SLF10 and to a lesser extent, SLF1, SLF3, SLF4 and SLF5 showed interaction with different S-RNase constructs. In addition, data in my study suggests that a bridge protein may be needed to stabilize proteins interactions between SLF and S-RNase. The work that has been completed will lead to a better understanding of self versus non-self recognition in pollination. An understanding of GSI mechanisms should also lead to the ability to manipulate breeding barriers in agricultural crops such as tomatoes, potatoes and fruit trees

Isolation and characterization of SLF genes from a BAC library representing the s-locus of Petunia axillaris

Gametophytic Self-incompatibility is the biochemical process in which plants can recognize their own (self) pollen, therefore rejecting it while accepting pollen of another plant (non-self) of the same species. This mechanism is governed by two genes that encode for pollen-recognition (pollen-S) and style-recognition (pistil-S) components which are more commonly known as SLF and S-RNase respectively. These genes are closely linked on the large, multiallelic S-locus and are, therefore, considered haplotypes of each other. When one of the SLF variants expressed in the haploid pollen matches the haplotype of the S-RNase in the diploid pistil tissue, rejection occurs through SR-Nase activity. If there is no match between the two haplotypes, SR-Nase is ubiquinated and the pollen can grow and fertilization occurs. In this research, SLF and S-RNase found in Petunia axillaris were studied to help gain an understanding in this process. One place to start is gathering more information on the S-Locus, since these genes are known to all be located there. It is estimated that the S-Locus, which is at least 8 MB in length, is known to house at least 118 genes. Although the organization of the S-locus is unique in that it is suppressed for recombination the entire S-locus has never been cloned or assembled. By establishing gene content, gene order, and linkage of the S-locus of P. axillaris, comparisons can be made between this self-compatible plant and self-incompatible plants of other species in the Solanaceae. This will aid in a better understanding of all the underlying mechanisms of GSI and will help in finding out how all the genes and their proteins involved in self-incompatibility interact at a molecular level. A combination of having the published genome for Petunia axillaris, together with a 5X BAC library for the sequenced line gives us the resources to assemble an entire S-locus for the first time. In this research, use of BAC library screening has determined that four different BAC clones house multiple SLF genes that show up on different scaffolds on the published genome. These results show that the scaffolds that house the SLF genes are near each other. In the future, these results can be used along with BAC-end sequencing and sequence alignment to the current P. axillaris genome to assemble a complete S-locus

Evolutionary Analysis of Basic RNase Genes from Rosaceous Species — S-RNase and Non-SRNase Genes

Over the past two and half decades there has been an explosion of progress in a growing number of model self incompatibility (SI) systems on our understanding of the molecular, biochemical and cellular processes underlying the recognition of self pollen and the initiation of a cascade of biochemical and cellular events that prevent self fertilization. These studies are unrevealing the complexity of a trait (SI) whose sole purpose, as far as we know, is to exert a strong influence on the breeding system of plants. Evolutionary interest in floral traits that influence the breeding system and in the forces that shape these traits began with Darwin who devoted one complete book to the subject (Darwin 1876) and significant portions of a second book. The evolution of plant breeding systems is often viewed as the interplay between the advantages and disadvantages of selfing. Evolutionary biologists have long noted that there are three primary advantages to selfing. First, there is an inherent genetic transmission advantage to selfing because a plant donates two haploid sets of chromosomes to each selfed seed and can still donate pollen to conspecifics. Second, selfing can provide reproductive assurance when pollinators are scarce or and third, it often costs less, in terms of energy and other resources, to produce selfed seed (e.g. fewer resources are expended to attract and reward pollinators. Some major questions remain unanswered concerning the evolution of stylar SRNases. Most pressing is the apparent disparity in patterns of diversification seen in the Solanaceae and Plantaginaceae relative to what is observed in the Rosaceae. Thus, we reviewing current publication regarding the evolutionary analysis basic RNases towards comprehensive view

Finding a Compatible Partner: Self-Incompatibility in European Pear (Pyrus communis); Molecular Control, Genetic Determination, and Impact on Fertilization and Fruit Set

Pyrus species display a gametophytic self-incompatibility (GSI) system that actively prevents fertilization by self-pollen. The GSI mechanism in Pyrus is genetically controlled by a single locus, i.e., the S-locus, which includes at least two polymorphic and strongly linked S-determinant genes: a pistil-expressed S-RNase gene and a number of pollen-expressed SFBB genes (S-locus F-Box Brothers). Both the molecular basis of the SI mechanism and its functional expression have been widely studied in many Rosaceae fruit tree species with a particular focus on the characterization of the elusive SFBB genes and S-RNase alleles of economically important cultivars. Here, we discuss recent advances in the understanding of GSI in Pyrus and provide new insights into the mechanisms of GSI breakdown leading to self-fertilization and fruit set. Molecular analysis of S-genes in several self-compatible Pyrus cultivars has revealed mutations in both pistil- or pollen-specific parts that cause breakdown of self-incompatibility. This has significantly contributed to our understanding of the molecular and genetic mechanisms that underpin self-incompatibility. Moreover, the existence and development of self-compatible mutants open new perspectives for pear production and breeding. In this framework, possible consequences of self-fertilization on fruit set, development, and quality in pear are also reviewed

Self-incompatibility in angiosperms

Samoinkompatibilnost je najvažniji mehanizam sprječavanja samooplodnje u kritosjemenjača. Genetički je kontrolirana najčešće jednim S-lokusom multialelnog tipa čija se ekspresija razlikuje unutar tkiva tučka i polena. Takvi tkivno specifični proteinski produkti istog S-alela stupaju u interakciju i pokreću mehanizam autosterilnosti. Najčešći sustavi SI-i su gametofitski (GSI) i sporofitski (SSI) sustav dok među ostale ubrajamo kasno ovarijsku (OSI) i kriptičnu samoinkompatibilnost (CSI). Detaljnije su proučena tri molekularna mehanizma: S-RNazni (Solanaceae tip), kalcijev kaskadni (Papaveraceae tip) i SSI homomorfnog tipa u porodici Brassicaceae. S-Rnazni mehanizam je najviše istraživani tip SI-a, no i dalje je upitan njegov točan način funkcioniranja. Iz svih informacija može se zaključiti kako su dva predložena modela najvjerojatnije povezana. Ubikvitinacijom se ostvaruje direktna degradacija ribonukleaza u citoplazmi dok se njihov endocitozni ulaz preusmjerava u vakuolu. Buduća istraživanja bi se trebala posvetiti predloženoj hipotezi kao i točnom mjestu citotoksične aktivnosti S-RNaza. Papaveraceae tip jedan je od najpotpunijih mehanizama gdje je većina interakcija poznata, a novootkrivena pojava indukcije SI odgovora u nesrodnim biljkama nudi nove mogućnosti u agronomskoj primjeni i genetičkim istraživanjima. Kroz ovaj rad prikazana je većina dosadašnjih saznanja u polju samoinkompatibilnosti te je uočljivo da mnogi segmenti tog polja čak i nakon 60 godina istraživanja ostaju neistraženi. Međudjelovanje više lokusa u jednosupnica, zanimljiva pojava heterostilije jaglaca i ostali sustavi SI-a svojim dubljim istraživanjem pružaju mogućnost važnih informacija za filogeniju, a time i detaljnije objašnjenje evolucijskih procesa koji su doprinijeli nevjerojatnoj divergenciji kritosjemenjača.Self-incompatibility is one of the most crucial mechanism to prevent inbreeding in angiosperms. It is genetically controlled with one multiallelic S-locus whose expression depends on pistil or pollen tissue. The most spread auto-sterility systems in angiosperms consists of two types of SI: gametophytically (GSI) or sporophytically (SSI) controlled self-incompatibility. The rest of SI noticed in angiosperms are cryptic (CSI) and late ovarian (OSI). Three molecular mechanisms have been closely observed: S-RNase system (present in Solanaceae), calcium cascade system (present in Papaveraceae) and homomorphic SI type in Brassicaceae family. After 60 years of intensive scientific research, our knowledge of molecular mechanisms of self-incompatibility is still incomplete. Even the most researched S-RNase mechanism remains questionable. From all of the information presented in this review, it can be concluded that the two proposed models are most likely related. Ubiquitination targets ribonucleases while most of them which enter the cell through endocytosis are sealed off from the cytoplasm in the vacuole. The most important suggestion for future research is to set the point of interest in connecting these two models and test this hypothesis, as well as look into the exact location of cytotoxic activities of S-RNases. Papaveraceae type is one of the most researched mechanisms and most of its interactions are well-known. The newly discovered phenomenon where scientists managed to induct SI in a completely unrelated plant species opens up new possibilities for agronomical application and genetic research. The focus of this paper is to deliver current information on the topic of self-incompatibility, even though most of SI mechanisms are still poorly documented in the majority of plant families. Deeper study of this problem would offer valuable information for phylogeny and better explanation of evolutionary processes of angiosperms

Genome editing in fruit, ornamental, and industrial crops

The advent of genome editing has opened new avenues for targeted trait enhancement in fruit, ornamental, industrial, and all specialty crops. In particular, CRISPR-based editing systems, derived from bacterial immune systems, have quickly become routinely used tools for research groups across the world seeking to edit plant genomes with a greater level of precision, higher efficiency, reduced off-target effects, and overall ease-of-use compared to ZFNs and TALENs. CRISPR systems have been applied successfully to a number of horticultural and industrial crops to enhance fruit ripening, increase stress tolerance, modify plant architecture, control the timing of flower development, and enhance the accumulation of desired metabolites, among other commercially-important traits. As editing technologies continue to advance, so too does the ability to generate improved crop varieties with non-transgenic modifications; in some crops, direct transgene-free edits have already been achieved, while in others, T-DNAs have successfully been segregated out through crossing. In addition to the potential to produce non-transgenic edited crops, and thereby circumvent regulatory impediments to the release of new, improved crop varieties, targeted gene editing can speed up trait improvement in crops with long juvenile phases, reducing inputs resulting in faster market introduction to the market. While many challenges remain regarding optimization of genome editing in ornamental, fruit, and industrial crops, the ongoing discovery of novel nucleases with niche specialties for engineering applications may form the basis for additional and potentially crop-specific editing strategies.The authors would like to acknowledge funding from MINECO, Spain (PGC2018-097655-B-I00 to P Christou), Generalitat de Catalunya Grant 2017 SGR 828 to the Agricultural Biotechnology and Bioeconomy Unit (ABBU). Work in the Dhingra lab in crop improvement is supported in part by Washington State University Agriculture Research Center Hatch grant WNP00011. ES and FR acknowledge the support received from the Department of Horticulture, BW was supported in part by a Research Assistantship from the Washington State University Graduate School. The authors would also like to thank Drs A. McHughen and H. Quemada for input and clarifications on US genome editing regulations. We would also like to thank the anonymous reviewers for their insightful comments

Sequence Characterization and Spatiotemporal Expression Patterns of PbS

Many flowering plants exhibit an important intraspecific reproductive barrier phenomenon, that is, self-incompatibility (SI), in which S-RNase genes play a significant role. To clarify the specific function of S-RNase genes in Chinese pears, the full length cDNA of PbS (26) -RNase was isolated by rapid amplification of cDNA ends (RACE) technology from Chinese white pear (Pyrus bretschneideri) cultivar “Hongpisu.” The cDNA sequence for PbS (26) -RNase was deposited in GenBank under accession number EU081888. At the amino acid level, the PbS (26) -RNase displayed the highest similarity (96.9%) with PcSa-RNase of P. communis, and only seven amino acid differences were present in the two S-RNases. Phylogenetic analysis of rosaceous S-RNases indicated that the PbS (26) -RNase clustered with maloideous S-RNases, forming a subfamily-specific not a species-specific group. The PbS (26) -RNase gene was specifically expressed in the style but not other tissues/organs. The expression level of the PbS (26) -RNase gene rapidly increased at bell balloon stage (BBS), and then it dropped after pollination. However, the abundance of the PbS (26) -RNase gene transcript in the style was greater after cross-pollination than after self-pollination. In addition, a method for rapidly detecting the PbS (26) -RNase gene was developed via allele-specific primers design. The present study could provide a scientific basis for fully clarifying the mechanism of pear SI at the molecular level