Periodic Reporting for period 1 - INTERSPREC (Investigation of interspecific pollen tube reception)
Reporting period: 2018-07-01 to 2020-06-30
As new species evolve during incipient speciation, different crossing barriers are formed, which prevent the formation of potentially unviable or sterile hybrid offspring. It has been considered that these barriers are weak at the beginning of the split of two species, still allowing the formation of hybrids, albeit this may be rare. Later, stronger barriers evolve that prevent any crossing and the two species are reproductively isolated.
We study how speciation emerges, looking at the origin of reproductive barriers. As tool we use two closely related species, Arabidopsis thaliana and Arabidopsis lyrata, where reproductive barriers are still in formation. Depending on the A.thaliana accession, A.lyrata pollen is recognized with a variable degree of success (between 10% and 90%). If recognition does not occur, the pollen tube (PT) is not properly received, keeps growing inside the ovule, and fertilization fails. This recognition step takes place in the cells at the contact point between the embryo sac and PT, the synergids, and can be considered the point of emergence of a first reproductive barrier. Genes involved in reproductive barriers are expected to show rapid divergence.
The work focuses on the regulation of the genes found to be important for interspecific PT recognition by the female gametophyte, as well as the molecular characterization of the recognition mechanism.
The proposed research project main objectives are:
1. Characterization of the newly discovered gene: generating additional alleles (CRISPR-Cas9), addressing redundancy in synergids, manipulation by ectopic expression
2. Identification of the promoter region of discovered gene from different Arabidopsis species
3. Identification of the factors regulating discovered gene in A. thaliana
4. Training, active dissemination, publication
We study how speciation emerges, looking at the origin of reproductive barriers. As tool we use two closely related species, Arabidopsis thaliana and Arabidopsis lyrata, where reproductive barriers are still in formation. Depending on the A.thaliana accession, A.lyrata pollen is recognized with a variable degree of success (between 10% and 90%). If recognition does not occur, the pollen tube (PT) is not properly received, keeps growing inside the ovule, and fertilization fails. This recognition step takes place in the cells at the contact point between the embryo sac and PT, the synergids, and can be considered the point of emergence of a first reproductive barrier. Genes involved in reproductive barriers are expected to show rapid divergence.
The work focuses on the regulation of the genes found to be important for interspecific PT recognition by the female gametophyte, as well as the molecular characterization of the recognition mechanism.
The proposed research project main objectives are:
1. Characterization of the newly discovered gene: generating additional alleles (CRISPR-Cas9), addressing redundancy in synergids, manipulation by ectopic expression
2. Identification of the promoter region of discovered gene from different Arabidopsis species
3. Identification of the factors regulating discovered gene in A. thaliana
4. Training, active dissemination, publication
In the first part of the project we proceeded to characterize the newly discovered gene.
To this aim we measure the expression of the gene of interest in the different accessions using digital droplet PCR (ddPCR) of gene of interest in A.thaliana. The result showed no quantifiable differences possibly due to the expression in only few cells of the ovule. Laser capture microdissection to extract specifically from synergids followed by deep sequencing could overcome this problem.
We then tested the role of the gene in analysis in the different accession by knocking out the gene using CRISPR-Cas9 technology. We indeed found that the gene is important for all the accessions in analysis for the pollen tube reception of pollen of different species (A.lyrata)
Ectopic expression of gene of interest in A.thaliana has been done with the promoter of the gene RPL18. Pollen tube overgrowth (PT-OG) phenotype in T2 lines has been carried out and showed that gene of interest overexpression is enough to decrease the PT-OG.
Complementation constructs (using units created before) has been generated. T2 generation plants confirmed that gene of interest can complement the phenotype of the mutant generated.
CRISPR-Cas9 construct for genes closely related to gene of interest has been made using newly published golden gate based system.
Mutant for LysMe2 single mutant showed no phenotype. Double mutant for LysMe2 and gene of interest showed no differences compared to mutant only for gene of interest. Mutant for LysMe1 showed possible gametophytic defect.
For the investigation of the most important region for the regulation of gene of interest, different sub-unit of the putative promoter has been analysed: the promoter, 3kb before ATG, gene (there are no introns), 1.4kb after stop from A.thaliana were used togheter with 3YFP fluorophore.
This region has been amplified from different varieties, then cloned with the gene of interest cds fused with CITRINE fluorophore. Different accessions have been transformed with the different constructs. The analysis did not show a particular difference in expression pattern.
Moreover, creation and test in plant of promoter subdivided in 6 part 500bp each found that 1,5kb region upstream the ATG is the minimal region for a correct expression. We also found that the region between 1,5kb and 1kb upstream the ATG is important for the restriction of the expression in the female gametophyte.
Finally to identify the factors regulating gene of interest in A. thaliana we performed a bioinformatic analysis of the region recognized in the previous experiments to be necessary for gametophyte regulation. We found three CArG boxes leading to the hypothesis that transcription factors binding CArG boxes (MADS-domain proteins) can be the regulatory factors of gene of interest. Yeast-one-hybrid (Y1H) assay is ongoing to confirm that this region is targeted by MADS-domain proteins. Versions of the 1.5kb promoter with mutations in the CarG boxes have been created and their expression will be tested in planta.
To this aim we measure the expression of the gene of interest in the different accessions using digital droplet PCR (ddPCR) of gene of interest in A.thaliana. The result showed no quantifiable differences possibly due to the expression in only few cells of the ovule. Laser capture microdissection to extract specifically from synergids followed by deep sequencing could overcome this problem.
We then tested the role of the gene in analysis in the different accession by knocking out the gene using CRISPR-Cas9 technology. We indeed found that the gene is important for all the accessions in analysis for the pollen tube reception of pollen of different species (A.lyrata)
Ectopic expression of gene of interest in A.thaliana has been done with the promoter of the gene RPL18. Pollen tube overgrowth (PT-OG) phenotype in T2 lines has been carried out and showed that gene of interest overexpression is enough to decrease the PT-OG.
Complementation constructs (using units created before) has been generated. T2 generation plants confirmed that gene of interest can complement the phenotype of the mutant generated.
CRISPR-Cas9 construct for genes closely related to gene of interest has been made using newly published golden gate based system.
Mutant for LysMe2 single mutant showed no phenotype. Double mutant for LysMe2 and gene of interest showed no differences compared to mutant only for gene of interest. Mutant for LysMe1 showed possible gametophytic defect.
For the investigation of the most important region for the regulation of gene of interest, different sub-unit of the putative promoter has been analysed: the promoter, 3kb before ATG, gene (there are no introns), 1.4kb after stop from A.thaliana were used togheter with 3YFP fluorophore.
This region has been amplified from different varieties, then cloned with the gene of interest cds fused with CITRINE fluorophore. Different accessions have been transformed with the different constructs. The analysis did not show a particular difference in expression pattern.
Moreover, creation and test in plant of promoter subdivided in 6 part 500bp each found that 1,5kb region upstream the ATG is the minimal region for a correct expression. We also found that the region between 1,5kb and 1kb upstream the ATG is important for the restriction of the expression in the female gametophyte.
Finally to identify the factors regulating gene of interest in A. thaliana we performed a bioinformatic analysis of the region recognized in the previous experiments to be necessary for gametophyte regulation. We found three CArG boxes leading to the hypothesis that transcription factors binding CArG boxes (MADS-domain proteins) can be the regulatory factors of gene of interest. Yeast-one-hybrid (Y1H) assay is ongoing to confirm that this region is targeted by MADS-domain proteins. Versions of the 1.5kb promoter with mutations in the CarG boxes have been created and their expression will be tested in planta.
The project represents a new line of research in a field that is in its beginning. To the best of our knowledge, unravelling the molecular mechanisms allowing the distinction of pollen from the same versus a different species during PT reception is a subject of study not covered by any other group.
At the same time, we are uncovering aspects related to the mechanisms of PT reception that can be translated for applications crop plants, where overcoming crossing barriers would open access to the tertiary gene pool.
We are also characterizing a group of genes that have not been studied so far. These genes are encoding proteins with single LYSM domain. The understanding of the function of protein with single LYSM domain has the potential to contribute to fields besides reproduction. In fact LYSM proteins have an important role in plant–pathogen interaction as well as in nodule formation nothing is known about the function of secreted protein with a single LYSM domain like the one in analysis.
At the same time, we are uncovering aspects related to the mechanisms of PT reception that can be translated for applications crop plants, where overcoming crossing barriers would open access to the tertiary gene pool.
We are also characterizing a group of genes that have not been studied so far. These genes are encoding proteins with single LYSM domain. The understanding of the function of protein with single LYSM domain has the potential to contribute to fields besides reproduction. In fact LYSM proteins have an important role in plant–pathogen interaction as well as in nodule formation nothing is known about the function of secreted protein with a single LYSM domain like the one in analysis.