UNDERSTANDING THE ROLE OF THE APHID MICROBIOME IN RUSSIAN WHEAT APHID VIRULENCE

The long-term goal of my research is to guide development of novel aphid control strategies through knowledge of how the RWA microbiome contributes to aphid virulence and/or biotypes. Here, I propose to (1) identify bacteria in the RWA microbiome that contribute to RWA virulence, (2) discover genes in these bacteria that encode candidates for virulence effectors and other functionally relevant proteins to aphid and/or plant interactions, and (3) use wheat differentials and RWA treated with synthetic bacterial communities (SynComs) to determine which bacteria contribute to virulence, and/or are responsible for RWA biotype. Understanding these interactions may lead to new management approaches targeted at disrupting the microbiome as means of RWA control.Obj. 1: Determine how the microbial community structure varies among RWA biotypes with different virulence patterns on resistant and susceptible wheat varieties. (1A) Characterize microbial communities associated with different RWA populations. (1B) Identify core microbiome and keystone species associated with RWA microbiome.Rationale: Biotypic differences in aphid-associated microbiomes is documented in sugarcane aphid and pea aphid, however, correlation of these differences with virulence has not been done[1, 2]. Analyzing community structure to detect differences in RWA microbial communities is a first step in the attempt to identify microbiota contributing to aphid virulence. Preliminary analysis of RWA amplicon sequencing data revealed unique taxa that are present in US2 and absent from US2-Clean. One of the abundant taxa identified in US2 and absent in US2-Clean is Erwinia iniecta, a microbe we found to be common to RWA[3, 4].Obj. 2: Evaluate the RWA microbiome functional profile and traits related to aphid virulence. (2A) Construct genomes and identify the functional potential of the RWA microbiome. (2B) Contrast metatranscriptomic profiles of biotypes to determine active microbe/RWA/plant interaction genes. Rationale: The genome of E. iniecta, a microbe associated with RWA biotype US2, contains two Type III Secretion Systems (TTSS). One TTSS is similar to that in the plant pathogen E. amylovora, and contains a putative effector, dspE, known to interact with plant R genes[5]. The second, a Salmonella-like TTSS, encodes Inv-Mxi-Spa type effectors required for persistence in insects[6]. Currently, we don't know the contribution of E. iniecta to the RWA microbial community, but its stable association with US2 populations (from lab reared and field samples) together with the genomic footprint of the bacterium make it a likely candidate for a key species in RWA virulence. E. iniecta does contain virulence factors, but this analysis doesn't represent the complexity that exists from a microbiome perspective. Thus, I will identify and catalog predicted functional effectors in the entire RWA bacterial community. First, a shotgun metagenomic approach will infer function of the microbiome and allow construction of metagenome assembled genomes (MAGs). Next, a transcriptomic approach will provide information on which bacteria are active in the community and will identify transcriptomic differences that correlate with aphid virulence, if any.Objective 3: Develop SynComs to identify bacteria responsible for enhanced virulence and to determine if bacteria are responsible for RWA biotype. (3A) Match living bacterial cells in RWA to bacteria of interest identified in Obj. 1&2. (3B) Construct SynComs of different bacterial composition. (3C) Evaluate aphid-SynCom-plant interactions.Rationale: To explore if RWA virulence patterns can be altered by modifying the aphid microbiome, SynComs of keystone microbes (Obj. 1) and microbes equipped with virulence factors (Obj. 2) will be added to RWA. These experiments would provide unequivocal evidence that the aphid uses bacterial effectors to interact with host R genes. The RWA microbiome is less complex than other explored systems (soils), so SynCom development should be straightforward. References Cited 1. Holt, J.R., et al., Differences in Microbiota between two Multilocus Lineages of the sugarcane Aphid (Melanaphis sacchari) in the continental United States. Annals of the Entomological Society of America, 2020.2. Gauthier, J.P., et al., Bacterial Communities Associated with Host-Adapted Populations of Pea Aphids Revealed by Deep Sequencing of 16S Ribosomal DNA. Plos One, 2015. 10(3).3. Campillo, T., et al., Erwinia iniecta sp. nov., isolated from Russian wheat aphid (Diuraphis noxia). International Journal of Systematic and Evolutionary Microbiology, 2015. 65(10): p. 3625-33.4. Luna, E., et al., Bacteria Associated with Russian wheat aphid (Diuraphis noxia) enhance aphid virulence to wheat. Phytobiomes, 2018. 2(3): p. 151-164.5. Bogdanove, A.J., D.W. Bauer, and S.V. Beer, <em>Erwinia amylovora</em> Secretes DspE, a Pathogenicity factor and functional AvrE homolog, through the Hrp (Type III Secretion) pathway. Journal of Bacteriology, 1998. 180(8): p. 2244-2247.6. Correa, V.R., et al., The bacterium Pantoea stewartii uses two different type III secretion systems to colonize its plant host and insect vector. Applied and Environmental Microbiology, 2012. 78(17): p. 6327-36.