EXPLORING THE ROLE OF PROPHAGES IN INTERFERING WITH THE MICROBIOME OF A PHYTOPLASMA INSECT VECTOR

ALTRO ABSTRACT Euscelidius variegatus (family Cicadellidae) is an insect vector of phytoplasmas, wall-less plant-pathogenic bacteria that cause yield losses in many crops worldwide. Phytoplasmas are obligate parasites colonizing the phloem of the host plant and the body of the insect vector. The main control strategies to limit these pathogens are insecticide treatments against the vector species. E. variegatus is a natural vector of chrysanthemum yellows phytoplasma, which is associated with a disease in ornamental plants in Northern Italy, and a laboratory vector of Flavescence dorée phytoplasma, which represents one of the major threats to grapevines, especially in Southern Europe. E. variegatus harbors an heritable bacterial endosymbiont, originally called BEV (Bacterial parasite of E. variegatus), which was recently assigned to the genus Symbiopectobacterium (family Enterobacteriaceae). BEV can be cultured in axenic conditions, but its growth is slow and its survival is limited in time. Some phage-like sequences were found in the metatranscriptome of E. variegatus and their association with BEV genome was assessed by PCR on BEV isolates growing in axenic cultures. Moreover, transmission electron microscopic (TEM) observations of phage particles, both in BEV cultures and in a viral purification obtained from E. variegatus, confirmed that some prophages are actively replicating. The objectives of this work are: i) to identify and characterize the prophages sequences harbored in the BEV genome; ii) to study prophages activation in different conditions; iii) to investigate the role of lysogenic phages in the survival of the phytoplasma vector E. variegatus. BEV genome was sequenced using Illumina and Nanopore technologies, assembled with Flye and polished with several rounds of Racon, Medaka and Pilon. Phaster program predicted 25 intact phage-like sequences in the 5 million bp-long BEV genome. All the predictions were manually checked for the presence of at least two structural proteins and eight bacteriophages belonging to the class Caudoviricetes were retained for further analyses. Preliminary results obtained with specific primers showed that five of them were active both in the insect and in the bacterial culture, one was active only in the bacterial culture and two appeared to be inactive in both conditions so far. Observations under a Philips CM10 TEM showed at least five different phage particle morphologies in both BEV cultures and insect body. After three days of growth in the dark at 26°C in Tryptic Soy Broth, BEV showed a slow linear growth. Such growth might be explained by a continuous, basal production of phage particles without any phage bursts. This phenomenon suggests that phages harboured in BEV genome are not true temperate phages and may have one of the alternative infection strategies (such as pseudo-lysogeny, carrier state, chronic infection) that are recently emerging as ubiquitous and potentially decisive in the ecology and evolution of their bacterial hosts. A BEV suspension was microinjected into an E. variegatus population lacking this endosymbiont. The survival of the injected insects reared on chrysanthemum decreased and the development of the new generation was delayed with respect to control. Our hypothesis is that in E. variegatus prophages could play a key role in maintaining the bacterial growth within levels tolerated by the insect host, whereas in non-host species/populations the lytic phase of prophages might not be triggered, thus causing an uncontrolled growth of the bacterium and the death of the insect host.