Exploration bioinformatique des interactions pollen–pistil chez Solanum chacoense

With 107 species distributed throughout Latin America, wild potatoes (Solanum sect. Petota) form an important germplasm reservoir for the breeding of their cultivated cousin (S. tuberosum). Yet, although they live in sympatry and have the physiological ability to hybridize with close relatives, those wild species exhibit strong reproductive isolation barriers. The preliminary results on which this thesis is based, presented in chapter 1, show that pollen-pistil interactions are at the center of this isolation: in a heterospecific pistil, pollen tubes (PTs) are slowed down and their receptivity to ovular chemoattractive signals is compromised.  As we will see in chapter 2, the review of the abundant existing scientific literature indicates that a large number of proteins expressed in pistils and PTs have been characterized in other species and that several contribute, through their interspecific divergence, to the establishment of specific checkpoints along the entire path of PTs in pistil. Among them, the LURE chemoattractive proteins controlling the directional guidance of PTs in Torenia and Arabidopsis are an outstanding example of molecular mediators of reproductive isolation. The objective of this thesis is to isolate candidate genes playing similar functions in tuber-bearing Solanaceous species. It is with this in mind that we have undertaken a large-scale exploration of the diversity of genes expressed in the ovules of S. chacoense, in an approach combining bioinformatics, proteomics and transcriptomics.  In chapter 3, we present a new sequence search tool, KAPPA, specifically dedicated to the detection of cystein-rich peptides (CRPs), a particular class of proteins that includes known ovular chemoattractants, as well as several other pollen–pistil interactants. This tool has facilitated the identification of new CRPs in the following chapters.  We detail the algorithm, present its main functions and provide results demonstrating its performance on calibrated datasets.  In chapter 4, we describe a study of the ovule secretome of S. chacoense comparing the protein content of exudates from mature ovules (which are able to attract PTs) and immature ovules (which are unable to do so). We present the tissue-free gravity-extraction method (tf-GEM) used to collect exudates and detail the bioinformatics analysis performed on mass spectrometry results. We review the 305 secreted proteins identified in ovule exudates, about half of which are regulated between the two conditions under study, analyze the associated functional categories, and discuss possible applications for the discovery of candidate genes.  Chapter 5 presents the results of a microarray analysis of the transcriptional response of S. chacoense ovaries to different pollination types: conspecific compatible, conspecific incompatible, and heterospecific compatible. We show that ovarian genes are activated or repressed at a distance as early as the first few hours after pollination and that this response is amplified and becomes specific to each male genotype as the PTs progress in the style until fertilization. We discuss the role of different mediators, including ethylene, in the long-range transmission of the pollination signal.  In chapter 6, we present the results of a deeper transcriptomic analysis performed by sequencing RNAs from PT guidance-competent (mature wild-type) ovules and PT guidance-incompetent (immature wild-type and embryo sac-devoid frk1 mutant) ovules in S. chacoense. We show how our hybrid method, relying on both de novo and ab initio assemblies against the S. chacoense genome published recently, led us to obtain a set of 4353 ovule-enriched transcripts. We detail the improvements made to existing genome annotations, including the discovery of new loci encoding reproductive CRPs. We present an analysis of the gene expression profile in the ovule, discuss associated functional categories, and conclude with the identification of a list of candidate chemoattractants.  Finally, chapter 7 details these candidate chemoattractants, the techniques used to express and purify them in bacteria and yeasts, and the microfluidic devices developed to verify their ability to attract the pollen tube. We include preliminary results obtained with this method and conclude on possible developments of our project.

Keyphrases: Solanum, Ovule, Chimioattraction, Wild potatoes, Reproductive isolation, Pollen tube guidance, Chemoattraction, Cysteine-rich proteins, Transcriptomics, Secretomics, Microfluidics