The association between overlooked microbial eukaryotes and plant holobiont: functionality and diversity

The plant holobiont is a relatively recent term in biology, introduced to address the assembly composed by the plant itself and its associated microbes. In order to address evolutionary selection within this frame, it becomes necessary to study plant-associated microbes, or microbiome, as a whole. A number of studies underpin the hypothesis that the picture of the plant microbiome is not yet complete in a number of aspects: from the role of abiotic factors in shaping microbial communities, to the whole microbial composition itself. The full comprehension of the microbiome scenario is, however, crucial to address the plant holobiont and therefore plant health. A still overlooked component of the plant microbiome, which I called overlooked microbial eukaryotes, includes a vast range of microbes, spanning from photoautotrophs to parasites, and has been proven to have prominent roles in other contexts such as soil or freshwaters. I applied ecological approaches as well as field experiments in order to address this overlooked component as a key of the still incomplete picture of the plant holobiont. Amplicon sequencing on natural A.thaliana populations from 15 sites in Germany, France, Spain, and Sweden, revealed that overlooked microbial eukaryotes are not occasional partners of the plant holobiont, both epiphytical and endophytical. On the contrary, they are able to widely interact with key hubs of the plant leaf microbiome such as Sphingomonas sp and the family of Caulobacteraceae, independently of the surrounding abiotic factors. Among others, microalgae have proven to be major shapers of microbial diversity for bacteria, fungi, and oomycetes. A newly established network analysis tool revealed that the presence of selected microalgae like Bracteacoccus sp. are linked to fluctuations of the pathogen Pseudomonas viridiflava, opening potential new fields in plant immunity research. The role of microalgae within the plant holobiont is also likely expressed by the symbiosis with lichenising fungi. A tight association between lichenising fungi and potential algal partners was found in shaping the leaf microbial diversity, prominently the epiphytic compartment. Amplicon sequencing on a time course-common garden experiment revealed that, beside microalgae, consumers like amoeboid organisms of the groups Lobosa, Conosa, Ciliophora also shape microbial diversity and follow specific succession patterns over time. XI Furthermore, the key role of overlooked microbial eukaryotes in the plant holobiont seems to be stable even at low concentrations. In fact, pools of low abundant overlooked microbial eukaryotes shape microbial diversity, to an extent never assessed before. In all these experiments, a key finding was the marginal effect of plant ecotype, as well as the marginal effect of latitudinal-climatic factors (with the exception of oomycetes), compared to the impact of overlooked microbial eukaryotes. My work gives novel insights into the ecology and the successions of overlooked microbial eukaryotes, revealing scenarios in which primary producers shape microbial diversity through the presence of single taxon such as Bracteacoccus sp, or together with lichenising fungi. They also influence microbial population through consumers and predators like Ciliophora and Lobosa, which undergo successions over time. My work further supports the autonomy of the assembly of microbial community from the plant host genetic background and from province related features, underpinning a core and multilevel role of overlooked microbial eukaryotes in the plant holobiont.