'Candidatus Phytoplasma solani': from insect transmission to complete genome sequencing of several strains inducing different symptoms in tomato

Members of the provisional genus ‘Candidatus Phytoplasma’ are obligate, intercellular prokaryotic parasites, restricted to the phloem sieve elements of plants and transmitted by phloem-feeding insects. Phytoplasmas induce distinct disease symptoms in infected plants, thus being interesting subjects for research on molecular plant-microbiome interactions. They possess a very small genome; however, the current unavailability of an axenic culture method and the low complexity of their genomes are obstacles in assembling phytoplasmas chromosomes. Bois noir (BN), the most widespread European grapevine yellows disease associated with ‘Candidatus Phytoplasma solani’, is regularly found in Italy including the vine growing region, Friuli Venezia Giulia. During the course of this three year study, we were able to transmit to the model plant tomato cv. Micro-Tom 9 ‘Candidatus Phytoplasma solani’ local strains from two ecological pathosystems involving the natural host plants, Urtica dioica and Convolvulus arvensis, and associated to different tuf-types of the pathogen, tuf-type a and tuf-type b respectively. Several investigations were carried out on the transmitted strains plus an additional strain already available at the University of Udine with the aim to investigate: (i) in planta colonization patterns, (ii) biological and molecular characterization of the strains and (iii) plant-pathogen interactions. We were able to distinguish our strains in three main clusters based on the analyses of tuf, secY, vmp1 and stamp gene sequences. It was possible to link tuf-type of the strains to specific symptoms appearance and to severity of ultrastructural modifications. Colonization pattern of four different ‘Ca. P. solani’ strains suggests that tuf-a derived strains tend to colonize the host slower than other strains. Additionally, combining MinION long-reads and Illumina short-reads sequencing we were able to obtain complete chromosome sequences of four ‘Ca. P. solani’ strains. Comparative genome analysis between these strains revealed impressive diversity considering they all belong to the same species. Chromosomes length ranged between 751.188 and 973.640 bp with annotated CDS varying between 725 and 1064. The four genome sequences were mined for the presence of proteins carrying signal peptides domain (ranging between 23 and 40), of which putative SP proteins with no known attribution (and therefore candidate effector genes) were ranging between 17 and 34. In particular, all strains lacked TENGU-like proteins, whereas proteins like SAP11, SAP54/PHIL1 and SAP05 were found. Comparisons with already known phytoplasma effector proteins and putative secreted proteins evidenced strain-specific sets of effectors that may explain the quite different symptoms induced on tomato. The fully assembled genome sequences are the first complete representatives of ‘Ca. P. solani’ species and contribute to provide the basis for studying pathogenicity-mechanisms and evolution of phytoplasmas, as well as of ‘Candidatus Phytoplasma solani’.