Fast Evolution and Lineage-Specific Gene Family Expansions of Aphid Salivary Effectors Driven by Interactions with Host-Plants

GBE Fast Evolution and Lineage-Specific Gene Family Expansions of Aphid Salivary Effectors Driven by Interactions with Host-Plants Helène Boulain1, Fabrice Legeai1,2, Endrick Guy1, Stephanie Morlière1, Nadine E. Douglas3,4, Jonghee Oh5, Marimuthu Murugan6, Michael Smith7, Julie Jaquiery1, Jean Peccoud8, Frank F. White9, James C. Carolan3, Jean-Christophe Simon1,†,*, and Akiko Sugio1,†,* 1 INRA, UMR1349, Institute of Genetics, Environment and Plant Protection, Le Rheu, France 2 Inria/IRISA GenScale, Campus de Beaulieu, Rennes, France 3 Department of Biology, Maynooth University, Maynooth, Co. Kildare, Ireland 4 UCD School of Biology and Environmental Science, University College Dublin, Dublin, Ireland 5 Department of Plant Pathology, Kansas State University, Manhattan, Kansas 6 Community Science College and Research Institute, Tamil Nadu Agricultural University, Madurai, India 7 Department of Entomology, Kansas State University, Manhattan, Kansas 8 UMR CNRS 7267 Ecologie et Biologie des Interactions, equipe Ecologie Evolution Symbiose, Universite de Poitiers, Poitiers, France 9 Department of Plant Pathology, University of Florida, Gainesville, Florida † These authors contributed equally to this work. *Corresponding authors: E-mails: ; . Accepted: May 16, 2018 Data deposition: The RNAseq data was submitted to NCBI SRA database and released on 19th April 2018 with the reference SRP14110. Abstract Effector proteins play crucial roles in plant-parasite interactions by suppressing plant defenses and hijacking plant physiological responses to facilitate parasite invasion and propagation. Although effector proteins have been characterized in many microbial plant pathogens, their nature and role in adaptation to host plants are largely unknown in insect herbivores. Aphids rely on salivary effector proteins injected into the host plants to promote phloem sap uptake. Therefore, gaining insight into the repertoire and evolution of aphid effectors is key to unveiling the mechanisms responsible for aphid virulence and host plant specialization. With this aim in mind, we assembled catalogues of putative effectors in the legume specialist aphid, Acyrthosiphon pisum, using transcriptomics and proteomics approaches. We identified 3,603 candidate effector genes predicted to be expressed in A. pisum salivary glands (SGs), and 740 of which displayed up-regulated expression in SGs in comparison to the alimentary tract. A search for orthologs in 17 arthropod genomes revealed that SG-up-regulated effector candidates of A. pisum are enriched in aphid-specific genes and tend to evolve faster compared with the whole gene set. We also found that a large fraction of proteins detected in the A. pisum saliva belonged to three gene families, of which certain members show evidence consistent with positive selection. Overall, this comprehensive analysis suggests that the large repertoire of effector candidates in A. pisum constitutes a source of novelties promoting plant adaptation to legumes. Key words: Acyrthosiphon pisum, salivary proteins, host adaptation, positive selection, pest evolution, plant defenses. Introduction Insects comprise the most diverse group of metazoans, and evidence indicates that the evolution of herbivory has played a fundamental role in promoting their species richness and diversification (Wiens et al. 2015). Almost half of the currently known insect species feed on plants (Wu and Baldwin 2010), ß The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an OpenAccess article distributedunder the terms ofthe Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/),whichpermits noncommercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact 1554 Genome Biol. Evol. 10(6):1554–1572. doi:10.1093/gbe/evy097 Advance Access publication May 18, 2018 GBE Evolution and Gene Family Expansions of Aphid Salivary Effectors and herbivorous insect groups exhibit faster rates of diversification compared with nonherbivorous species (Wiens et al. 2015). A central hypothesis accounting for higher species richness in herbivorous insects proposes an evolutionary interaction between plant defense mechanisms and plant exploitation strategies of insects (Janz 2011). Furthermore, continuous interactions between host plants and herbivorous insects are predicted to make herbivore generalism difficult and constrain a given insect species to one or a few host species (Forister et al. 2015). Since plants provide not only food resources, but also habitats and mating sites to many herbivorous insects, plant specialization may induce divergent selection in insect populations at a range of traits that can lead to reproductive isolation and speciation (Peccoud et al. 2010; Mullen and Shaw 2014). Attempting to unveil the basic mechanisms of insect herbivory provides opportunities to understand the evolutionary and mechanistic basis of plant specialization by herbivorous insects, in particular, and the diversification of metazoan life, in general. Aphids (Insecta: Aphidomorpha) are pests of wild and cultivated plants that directly reduce plant nutrients by ingesting phloem sap and indirectly cause diseases by transmitting plant pathogens (Blackman and Eastop 2000; Harris and Maramorosch 2014). Aphids are also excellent subjects for host specialization studies. Their clade is composed of approximately 5,000 species (Blackman and Eastop 2000), and most are considered to be plant specialists (Peccoud et al. 2010). Aphid mouthparts are modified into a rostrum or beak with the mandibles and maxillae forming needle-like stylets. Aphids secrete gelling saliva during the early stages of feeding to form a feeding sheath surrounding the stylets, and then secrete watery saliva into various plant cells (Moreno et al. 2011). Saliva contains effectors that modulate physiological responses to herbivory and permit feeding (Rodriguez et al. 2017). These effectors are likely exposed to natural selection, in particular by plant surveillance systems and defense mechanisms (Will et al. 2013). Interference with plant defenses through various mechanisms has been demonstrated for several effectors secreted by microbial plant pathogens, which ultimately promotes persistence and even spread of these pathogens (Varden et al. 2017). A subset of effectors, the so-called avirulence proteins, are detected by plant surveillance systems and trigger strong immunity in specific plants, determining the incompatibility (Bent and Mackey 2007). Effector genes are diverse, making prediction of effector functions often difficult from the amino acid sequences. As a result, relatively few salivary effectors from aphids have characterized interactions with active host defense responses. In planta expression of salivary (...truncated)