• RNA-seq
• retrotransposons
• mycorrhizal
• sunflower

Arbuscular mycorrhizal (AM) symbiosis is a beneficial interaction between plant and fungus occurring in almost 80% of land plants, AM fungi can penetrate the root creating a tree-shaped structure called arbuscule within the host cell. Fungus eases host nutrients uptake from the soil, especially phosphorus (P) and nitrogen (N), in exchange it receives sugars deriving from plant photosynthesis. AM symbiosis in agriculture reduce the impact of fertilizer for crops.
Sunflower (Helianthus annuus L.) is a worldwide cultivated oilseed crop belonging to the asteraceae family, the most numerous amongst angiosperms. Recently has been assessed its mycorrhizal status in wild and cultivated genotypes but no molecular data are available for symbiosis in this plant. H.annuus genome (inbred line XRQ) has been recently sequenced and represents a complex challenge due to its size (about 3.3 Giga bp) and composition, made of 80% of repetitive elements represented especially by retrotransposons.
Next generation sequencing RNA-seq allows exploration of wide transcriptome changes in cells or tissues under altered status as for instance during plant-fungus interaction. This method has been exploited to asses gene expression during AM symbiosis only in leguminosae and solanaceae but barely is known for other species.
Aim of this work is to exploit RNA-seq approach on mycorrhizal roots of sunflower during early and late phase of symbiosis in order to investigate both gene and retrotransposons expression.
For the experimental procedures several seeds of H.annuus (inbred line HA412-HO) were germinated then one-week old plantlets were split in two groups, one of control and the other inoculated with the AM fungus Rhizoglomus irregulare. In order to establish symbiosis progression staining analyses have been performed on inoculated roots. Roots of control and mycorrhizal plantlets were harvested at 4 and 16 days post inoculation, representing early and late stage of symbiosis,respectively. Three biological replicates for treatment and harvest point were retained and mRNA extracted from harvested roots. Overall 12 cDNA libraries were sequenced by Illumina sequencer.
In the first part of the thesis sequence reads deriving from the libraries were aligned on sunflower transcriptome reference (inbred line XRQ). Raw counts were normalized and statistical analyses on pairwise comparison between control and inoculated roots at early and late stage of colonization were carried out to identify differentially expressed genes (DEGs). Overall 19 up-regulated DEGs were found during the early phase of symbiosis while 694 and 13 respectively up and down-regulated transcripts have been retrieved in the late stage of colonization. Whole DEGs detected during early phase were differentially expressed also in the late phase of symbiosis suggesting a pivotal role of these genes for onset and maintenance of plant-fungus interaction. Transcripts differentially expressed during late phase of symbiosis were principally involved in biological functions such as signaling, cell-wall and membrane remodeling, transcription, hormone biosynthesis and transport. Besides known genes related to symbiosis even some brand new transcripts were detected in plant-fungus interaction as for instance encoding several BAHD-acyltransferases, an oxoglutarate dependent dioxygenase and a stellacyanin-like protein.
In the second part of the thesis, in order to analyse retrotransposon expression during AM symbiosis, reads from the libraries were mapped on reverse transcriptase (RT) retrotranspons domain sequences extracted from whole genome assembly of sunflower inbred line HA412-HO. Corresponding expression values of raw counted reads were analyzed in order to investigate RT expression in mycorrhizal and control libraries at early and late phase of symbiosis. Globally 46 RTs, mostly belonging to Copia superfamily, resulted expressed in control and inoculated samples. Amongst these 4 Copia were differentially expressed between control and inoculated roots after 16 days of symbiosis. In addition, at genomic sequence level, expressed RTs showed a significant higher number of genes placed in the proximity (100,000 bp up and down-stream) of the sequence compared to not expressed ones, suggesting that RT expression could be related to epigenetic control of gene expression. Finally, considering the retrotranpsosons activation after environmental stimuli, the expression of the 46 RTs was compared to other biotic (GA3, SA, IAA, Strigolactones, Brassinosteroids, Ethylene, Kinetin) and abiotic (Salt, PEG) stimuli on sunflower roots. After comparison 25 RTs were found to be expressed in at least one of the treatments, 17 were expressed specifically during fungal symbiosis and 4 were differentially expressed uniquely after plant-fungus interaction.
In conclusion AM symbiosis between H. annuus and R. Irregulare leads to a wide remodeling of plant transcriptome concerning both genes and non-genes sequences such as retrotransposons, that may possibly cause rearrangement of host genome.