A Functional Genomics Approach Identifies Candidate Effectors from the Aphid Species Myzus persicae (Green Peach Aphid)

et al. (2010) A Functional Genomics Approach Identifies Candidate Effectors from the Aphid Species Myzus persicae (Green Peach Aphid). PLoS Genet 6(11): e1001216. doi:10.1371/journal.pgen.1001216 A Functional Genomics Approach Identifies Candidate Effectors from the Aphid Species Myzus persicae (Green Peach Aphid) Jorunn I. B. Bos 0 David Prince 0 Marco Pitino 0 Massimo E. Maffei 0 Joe Win 0 Saskia A. Hogenhout 0 Gregory P. Copenhaver, The University of North Carolina at Chapel Hill, United States of America 0 1 Department of Disease and Stress Biology, The John Innes Centre , Norwich , United Kingdom , 2 Plant Physiology Unit, Department of Plant Biology and Centre of Excellence CEBIOVEM, University of Turin , Turin, Italy, 3 The Sainsbury Laboratory, Norwich , United Kingdom Aphids are amongst the most devastating sap-feeding insects of plants. Like most plant parasites, aphids require intimate associations with their host plants to gain access to nutrients. Aphid feeding induces responses such as clogging of phloem sieve elements and callose formation, which are suppressed by unknown molecules, probably proteins, in aphid saliva. Therefore, it is likely that aphids, like plant pathogens, deliver proteins (effectors) inside their hosts to modulate host cell processes, suppress plant defenses, and promote infestation. We exploited publicly available aphid salivary gland expressed sequence tags (ESTs) to apply a functional genomics approach for identification of candidate effectors from Myzus persicae (green peach aphid), based on common features of plant pathogen effectors. A total of 48 effector candidates were identified, cloned, and subjected to transient overexpression in Nicotiana benthamiana to assay for elicitation of a phenotype, suppression of the Pathogen-Associated Molecular Pattern (PAMP)-mediated oxidative burst, and effects on aphid reproductive performance. We identified one candidate effector, Mp10, which specifically induced chlorosis and local cell death in N. benthamiana and conferred avirulence to recombinant Potato virus X (PVX) expressing Mp10, PVX-Mp10, in N. tabacum, indicating that this protein may trigger plant defenses. The ubiquitin-ligase associated protein SGT1 was required for the Mp10-mediated chlorosis response in N. benthamiana. Mp10 also suppressed the oxidative burst induced by flg22, but not by chitin. Aphid fecundity assays revealed that in planta overexpression of Mp10 and Mp42 reduced aphid fecundity, whereas another effector candidate, MpC002, enhanced aphid fecundity. Thus, these results suggest that, although Mp10 suppresses flg22-triggered immunity, it triggers a defense response, resulting in an overall decrease in aphid performance in the fecundity assays. Overall, we identified aphid salivary proteins that share features with plant pathogen effectors and therefore may function as aphid effectors by perturbing host cellular processes. - Funding: This work was funded by Marie Curie International Reintegration Grant PIRG-GA-2008-230995 awarded to JIBB and The John Innes Centre (JIC). SAH is funded by the JIC and The Gatsby Charitable Foundation, DP by a studentship from the Biotechnology and Biological Sciences Research Council (BBSRC), MP by a grant from the Doctorate School of Science and High Technology of the University of Turin, Italy, and JW by the Gatsby Charitable Foundation. JIC is grant-aided by the BBSRC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Like most plant parasites, aphids require intimate associations with their host plants to gain access to nutrients. Aphids predominantly feed from the plant phloem sieve elements, and use their stylets to navigate between the cells of different layers of leaf tissue during which plant defenses may be triggered. Indeed, aphid feeding induces responses such as clogging of phloem sieve elements and callose formation, which are suppressed by the aphid in successful interactions with plant hosts [1]. In addition, some aphid species can alter host plant phenotypes, by for example inducing the formation of galls or causing leaf curling [2] indicating that there is an active interplay between host and aphid at the molecular level. During probing and feeding, aphids secrete two types of saliva: gelling saliva, which is thought to protect stylets during penetration, and watery saliva, which is secreted into various plant host cell types and the phloem [3]. The secretion of aphid saliva directly into the host-stylet interface [4], suggests that molecules present in the saliva may perturb plant cellular processes while aphids progress through different feeding stages. Interestingly, the knock-down of the C002 salivary gene in Acyrthosiphon pisum (pea aphid) negatively impacts survival rates of this aphid on plant hosts [5,6]. Furthermore, proteomics studies based on artificial aphid diets showed the presence of secreted proteins, including C002, in aphid saliva indicating that these proteins are delivered inside the host plant during feeding [7,8]. However, whether and how these aphid salivary proteins function in the plant host remains elusive. Suppression of host defenses and altering host plant phenotypes is common in plant-pathogen interactions and involves secretion of molecules (effectors) that modulate host cell processes [9,10]. Therefore it is likely that aphids, similar to plant pathogens, deliver effectors inside their hosts to manipulate host cell process enabling successful infestation of plants [9]. Effector-mediated suppression Aphids are insects that can induce feeding damage, achieve high population densities, and most importantly, transmit economically important plant diseases worldwide. To develop durable approaches to control aphids, it is critical to understand how aphids interact with plants at the molecular level. Aphid feeding induces plant defenses, which can be suppressed by aphid saliva. Thus, aphids can alter plant cellular processes to promote infestation of plants. Suppression of plant defenses is common in plant pathogens and involves secretion of effector proteins that modulate host cell processes. Evidence suggests that aphids, like plant pathogens, deliver effectors inside their host cells to promote infestation. However, the identity of these effectors and their functions remain elusive. Here, we report a novel approach based on a combination of bioinformatics and functional assays to identify candidate effectors from the aphid species Myzus persicae. Using this approach, we identified three candidate effectors that affect plant defense responses and/or aphid reproductive performance. Further characterization of these candidates promises to reveal new insights into the plant cellular processes targeted by aphids. of plant defenses, such as Pathogen-Associated Molecular Patter (...truncated)