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From male-killers to plant pathogens: Investigation of cross-kingdom host-symbiont interactions in the Arsenophonus clade”

Periodic Reporting for period 1 - PHYTOPHONUS (From male-killers to plant pathogens: Investigation of cross-kingdom host-symbiont interactions in the Arsenophonus clade”)

Reporting period: 2018-09-01 to 2020-08-31

Bacterial symbionts are ubiquitous and influential partners of all metazoan life forms, including humans. In insects, this is exemplified by a high number of species harbouring heritable bacterial symbionts which provide important services to their hosts (e.g. nutrients or protection against pathogens). After millions of years of co-evolution, these bacteria are specifically adapted to living inside their host and often can no longer survive on their own. In addition, numerous insect species are vectors of plant-pathogenic bacteria, which cause devastating crop diseases and important economic losses. The insect vectors are generally hemipteran insects (e.g. leafhoppers, planthoppers or psyllids), which acquire and transmit the bacteria while feeding on plants. It is thus evident that certain bacteria have evolved adaptations to very different host environments from two kingdoms of life and are able to switch frequently between these contrasting lifestyles. However, a detailed understanding of the adaptations allowing an insect symbiont to become a plant pathogen is still lacking, due to the scarcity of bacterial model organisms at the early stages of transition from a purely insect-associated to a multi-host lifestyle.
The project PHYTOPHONUS investigated the genomic adaptations allowing an initially insect-associated bacterial symbiont to successfully switch between insect and plant hosts and to become a plant pathogen. This was achieved using the phytopathogenic strain ‘Candidatus Phlomobacter fragariae’ belonging to the widespread Arsenophonus clade of insect symbionts as a model system. Despite being firmly established as insect symbionts, two different strains belonging to the clade have become insect-vectored plant pathogens, causing either Marginal Chlorosis Disease of strawberry or the disease “basses richesses” (=low sugar content) of sugar beet. In both cases, the bacteria are vectored by planthoppers and accumulate in the plant phloem, ultimately causing yellows, necrosis and plant death. Considering that both diseases first appeared less than 30 years ago and to date have only been observed in restricted and disconnected localities (France, Italy and Japan), it can be assumed (a) that the switch from an ancestral, purely insect-associated, to a multi-host lifestyle occurred very recently and (b) that it occurred independently multiple times within the Arsenophonus clade. These symbionts therefore represent outstanding model systems to investigate bacterial adaptations and cross-kingdom host interactions at the early stages of transition towards an insect-vectored phytopathogen.

The specific objectives within this project were:
(i) to test whether the ability of ‘Ca. P. fragariae’ to infect plant tissues was achieved via genomic adaptations compared to other Arsenophonus strains or whether the genetic repertoire of this clade is sufficiently versatile to allow rapid adaptations to plant hosts;
(ii) to investigate the symbiotic interactions of ‘Ca. P. fragariae’ with both its insect and plant hosts and their respective microbiomes, thereby providing a holistic picture of this multi-partite relationship.
We first investigated whether a non-phytopathogenic strain of the Arsenophonus clade was able to survive in plants if forced to do so. This was achieved through experimental infections of plants with Arsenophonus nasoniae, the male-killing symbiont of the parasitoid wasp Nasonia vitripennis. Experimental infections of fava bean (Vicia faba) and periwinkle (Catharanthus roseus) were performed using different infection methods: (a) Using the leafhopper Euscelidius variegatus injected with the bacterium as experimental insect vector, and (b) through a series of direct plant inoculations (injections, grafting, immersion in bacterial solution). The results demonstrate that the male-killing strain A. nasoniae can easily infect another insect species, which was then able to transmit the bacterium to its host plants with a transmission rate of up to 30%. However, A. nasoniae did not establish stable infections of the plants and did not cause any disease symptoms, indicating that this Arsenophonus strain does not thrive in a plant host environment. This reinforced the hypothesis that genomic adaptations may have occurred in the phytopathogenic strains of this clade. To verify this hypothesis, we produced high-quality draft genomes of both phytopathogenic Arsenophonus strains, ‘Ca. Phlomobacter fragariae’ and ‘Ca. Arsenophonus phytopathogenicus’, using Illumina sequencing. These genomes are now being compared to the genomes of other Arsenophonus symbionts to identify genomic adaptations linked to phytopathogenicity.
Furthermore, we analysed the microbiomes of ‘Ca. P. fragariae’-infected and uninfected insects as well as of infected and uninfected strawberry plants using amplicon sequencing of the 16S rRNA gene. The aim of this approach was two-fold: (i) Investigate whether ‘Ca. P. fragariae’ impacts microbiome composition of both its insect vector and plant host and (ii) identify potential bacterial antagonists within the natural bacterial communities of both host organisms. The results indicate that the insect vector harbours a very simple microbiome, whereas the microbiome of strawberry is much more diverse. An in-depth analysis of microbial interactions within this diverse bacterial community is currently ongoing, with the aim to identify candidate bacterial taxa that might compete with ‘Ca. P. fragariae’ and that could be tested as biological control agents.
We also discovered a new transmission mechanism of ‘Ca. P. fragariae’ between strawberry plants. Strawberry plants can produce clonal daughter plants through stolons. We demonstrated that ‘Ca. P. fragariae’ is efficiently transmitted to the young plants through stolons and that bacterial loads in the young plants rapidly reach those of the mother plant, accompanied by disease symptoms. This finding may be of importance for the strawberry production sector, as stolon transmission could contribute to spreading the disease, unless symptoms develop quickly enough for the infected plants to be eliminated.
Our results demonstrate that the capacity to establish infections and to cause disease in plants is not ubiquitous in the Arsenophonus clade, suggesting specific adaptations in the phytopathogenic strains. From an evolutionary perspective, this project therefore contributes insights into the genomic adaptations allowing an insect-associated bacterial symbiont to become a plant pathogen. Furthermore, PHYTOPHONUS provided a holistic view of all the partners involved in this system: The bacterial pathogen, its insect vector and host plant, and their respective microbiomes. This approach produced new knowledge that may prove useful for the integrated management of Marginal Chlorosis Disease of Strawberry. Notably, the stolon transmission of the pathogen will be of direct importance for strawberry producers affected by this disease in the Dordogne region in France. Moreover, the comprehensive microbiome dataset of strawberry plants represents a valuable resource for the identification and testing of potential bacterial antagonists as biological control agents. Similarly, the genomes of the two phytopathogenic bacteria will inform on candidate bacterial effectors involved in the association with the insect vector and host plant, potentially leading to new pest control strategies.