Final Report Summary - ARABIDOPSI HYBRIDS (Analysis of arabidopsis hybrid incompatibilities)
While the discovery and large-scale exploitation of hybrid vigor was one of the most important breakthroughs in modern agriculture, failure of many wide crosses limits the beneficial traits that can be introduced into crops. Our Marie Curie IEF project 'Analysis of Arabidopsis hybrid incompatibilities' has consisted of two subprojects with the goal of understanding the basis of hybrid incompatibility within, and between, species.
For one sub-project, I have studied a hybrid between Arabidopsis thaliana accessions, Bla-1 and Sha, where plants have an abnormal appearance and reduced biomass. I identified a receptor-like protein kinase (RLK; Outgrowth-Associated Kinase (OAK)) that causes the plants to develop in this irregular manner. Causality was confirmed by adding a copy of the gene encoding this protein back into plants and recovering plants with a similar appearance to the hybrids. Removing the kinase activity of the protein also abrogates the phenotypic effects. The OAK gene is not present in the reference accession for Arabidopsis thaliana but has been formed by gene duplication and subsequent sequence divergence. A survey of 90 accessions showed that OAK is present in approximately one third of accessions.
The Arabidopsis thaliana genome encodes over 600 RLKs, and RLK genes exhibit exceptionally high within-species sequence variation. This family has been implicated in root and shoot development, in disease resistance and in plant-microbe interactions, all vitally important plant traits for agriculture, yet the function and ligands of most plant RLKs are still unknown.
RLKs often function as dimers, and consist of an extra-cellular domain that perceives a signal, and an intracellular kinase domain that activates a downstream signalling cascade. An incompatibility can be caused by changes in gene expression, changes in protein sequence, or a combination of both. For OAK, I found that the expression domain of the protein is important as are changes within the extracellular domain. I used a reporter assay to show that the equivalent RLK gene in the reference accession is expressed in the leaf blade, while OAK is expressed in the leaf stems. The extracellular domain is particularly variable between OAK in Bla-1 and Sha as compared to the kinase domain, therefore changes in the OAK extracellular domain may lead to perception of a new signal or hyper-activation of the protein. Genome-wide gene expression in the hybrid plants was compared to the parents, with the expectation that disease-resistance pathways may be up-regulated as in other hybrid incompatibilities. However, no pattern in the changes in expression of specific gene ontology categories could be found. This work continues with experiments underway to identify proteins downstream of OAK in the signalling cascade.
A second sub-project involves the genome-wide characterisation of crosses within and between species. A class of molecules called small RNAs (sRNAs) that regulate gene expression are maternally inherited. Therefore I speculated that changes in small RNA populations in hybrids may contribute to mis-regulation of genes and hence result in hybrid incompatibility. I examined sRNAs and genome-wide gene expression in F1 hybrids between A. thaliana accessions that are compatible and also in a hybrid between A. thaliana and A. lyrata that results in infertile F1 plants.
sRNA data and gene-expression information has been collected from all parents and hybrids of crosses involved in this study. Bioinformatic analysis of the data has been challenging due to the involvement of genomes that have not been fully sequenced. Intraspecific crosses are being utilised to examine the effect of structural variations and polymorphisms on gene expression in different accessions and their hybrids.
Data has been shared with other research groups to generate the maximum benefit from the project.
For one sub-project, I have studied a hybrid between Arabidopsis thaliana accessions, Bla-1 and Sha, where plants have an abnormal appearance and reduced biomass. I identified a receptor-like protein kinase (RLK; Outgrowth-Associated Kinase (OAK)) that causes the plants to develop in this irregular manner. Causality was confirmed by adding a copy of the gene encoding this protein back into plants and recovering plants with a similar appearance to the hybrids. Removing the kinase activity of the protein also abrogates the phenotypic effects. The OAK gene is not present in the reference accession for Arabidopsis thaliana but has been formed by gene duplication and subsequent sequence divergence. A survey of 90 accessions showed that OAK is present in approximately one third of accessions.
The Arabidopsis thaliana genome encodes over 600 RLKs, and RLK genes exhibit exceptionally high within-species sequence variation. This family has been implicated in root and shoot development, in disease resistance and in plant-microbe interactions, all vitally important plant traits for agriculture, yet the function and ligands of most plant RLKs are still unknown.
RLKs often function as dimers, and consist of an extra-cellular domain that perceives a signal, and an intracellular kinase domain that activates a downstream signalling cascade. An incompatibility can be caused by changes in gene expression, changes in protein sequence, or a combination of both. For OAK, I found that the expression domain of the protein is important as are changes within the extracellular domain. I used a reporter assay to show that the equivalent RLK gene in the reference accession is expressed in the leaf blade, while OAK is expressed in the leaf stems. The extracellular domain is particularly variable between OAK in Bla-1 and Sha as compared to the kinase domain, therefore changes in the OAK extracellular domain may lead to perception of a new signal or hyper-activation of the protein. Genome-wide gene expression in the hybrid plants was compared to the parents, with the expectation that disease-resistance pathways may be up-regulated as in other hybrid incompatibilities. However, no pattern in the changes in expression of specific gene ontology categories could be found. This work continues with experiments underway to identify proteins downstream of OAK in the signalling cascade.
A second sub-project involves the genome-wide characterisation of crosses within and between species. A class of molecules called small RNAs (sRNAs) that regulate gene expression are maternally inherited. Therefore I speculated that changes in small RNA populations in hybrids may contribute to mis-regulation of genes and hence result in hybrid incompatibility. I examined sRNAs and genome-wide gene expression in F1 hybrids between A. thaliana accessions that are compatible and also in a hybrid between A. thaliana and A. lyrata that results in infertile F1 plants.
sRNA data and gene-expression information has been collected from all parents and hybrids of crosses involved in this study. Bioinformatic analysis of the data has been challenging due to the involvement of genomes that have not been fully sequenced. Intraspecific crosses are being utilised to examine the effect of structural variations and polymorphisms on gene expression in different accessions and their hybrids.
Data has been shared with other research groups to generate the maximum benefit from the project.