Focus on Ethylene: Disruption of Ethylene Responses by Turnip mosaic virus Mediates Suppression of Plant Defense against the Green Peach Aphid Vector

Plants employ diverse responses mediated by phytohormones to defend themselves against pathogens and herbivores. Adapted pathogens and herbivores often manipulate these responses to their benefit. Previously, we demonstrated that Turnip mosaic virus (TuMV) infection suppresses callose deposition, an important plant defense induced in response to feeding by its aphid vector, the green peach aphid (Myzus persicae), and increases aphid fecundity compared with uninfected control plants. Further, we determined that production of a single TuMV protein, Nuclear Inclusion a-Protease (NIa-Pro) domain, was responsible for changes in host plant physiology and increased green peach aphid reproduction. To characterize the underlying molecular mechanisms of this phenomenon, we examined the role of three phytohormone signaling pathways, jasmonic acid, salicylic acid, and ethylene (ET), in TuMV-infected Arabidopsis (Arabidopsis thaliana), with or without aphid herbivory. Experiments with Arabidopsis mutants ethylene insensitive2 and ethylene response1, and chemical inhibitors of ET synthesis and perception (aminoethoxyvinyl-glycine and 1-methylcyclopropene, respectively), show that the ET signaling pathway is required for TuMV-mediated suppression of Arabidopsis resistance to the green peach aphid. Additionally, transgenic expression of NIa-Pro in Arabidopsis alters ET responses and suppresses aphid-induced callose formation in an ET-dependent manner. Thus, disruption of ET responses in plants is an additional function of NIa-Pro, a highly conserved potyvirus protein. Virus-induced changes in ET responses may mediate vector-plant interactions more broadly and thus represent a conserved mechanism for increasing transmission by insect vectors across generations.Plants suffer from numerous pathogen and herbivore challenges in both natural and agricultural environments, often facing multiple simultaneous threats (Casteel and Hansen, 2014). For example, many plant pathogens depend on insect vectors for transmission, including over 75% of all described plant viruses (Nault, 1997). Thus, plants must recognize, prioritize, and mount the most appropriate response to both the insect that is feeding and the pathogen being transmitted. Despite constant attack, plants persist, largely due to a sophisticated surveillance system. Plants respond with an arsenal of defenses that may be morphological, biochemical, or molecular in nature (Jones and Dangl, 2006; Jander and Howe, 2008). Nevertheless, pathogens and insects successfully colonize plants by actively compromising plant perception and/or defense responses.Recent studies show that synergisms exist between challengers, where both parties benefit during dual attack. For example, some virus infections can decrease plant defenses against insects, increasing plant palatability and vector fitness. Consequently, improved insect performance will increase the number of viruliferous vectors, promoting virus transmission to new hosts (Mauck et al., 2010; Casteel and Jander, 2013; Casteel et al., 2014; Li et al., 2014). Thus, vector-plant interactions represent a critical and synergistic relationship, ultimately determining survival and host range. Although numerous studies have examined virus-plant interactions, few have examined the molecular and genetic mechanisms mediating plant-virus-vector interactions and alterations in plant defenses (Li et al., 2014; Mauck et al., 2014).While defenses vary widely across plant species, the phytohormones that regulate their production are somewhat conserved. Modulation of hormone composition, timing, and concentration specifies plant responses to an attack (Mur et al., 2006; Verhage et al., 2010) and represents an excellent target for compromising defenses. Numerous studies have demonstrated that at least three phytohormones, jasmonic acid (JA), salicylic acid (SA), and ethylene (ET), have major roles in orchestrating plant defense responses (Bari and Jones, 2009; Erb et al., 2012; Pieterse et al., 2012). In general, SA signaling is critical for defense responses against a wide range of pathogens, including viruses (Glazebrook, 2005; Carr et al., 2010). Production of JA and ET, meanwhile, are involved in regulation of plant response to herbivores, necrotrophic pathogens, and nonpathogenic microbes (Glazebrook, 2005; Howe and Jander, 2008; Van der Ent et al., 2009). Virus infection can also alter JA and ET signaling (Carr et al., 2010; Lewsey et al., 2010; Wei et al., 2010; Mauck et al., 2014).Together, Arabidopsis (Arabidopsis thaliana), the green peach aphid (Myzus persicae), and Turnip mosaic virus (TuMV) constitute an excellent model system for investigating the molecular and biochemical mechanisms that underlie plant-aphid-virus interactions. As a well-studied model plant, Arabidopsis provides numerous genetic resources that can be used to investigate responses to aphid feeding and virus infection. The green peach aphid is a broad-host-range aphid and the world’s most prolific plant virus vector, transmitting more than 100 different viral species (Kennedy et al., 1962). The green peach aphid is the most common aphid pest on Arabidopsis in greenhouses and growth chambers (Bush et al., 2006), and we also have observed it feeding from Arabidopsis growing in nature. Due to the agricultural relevance of the green peach aphid, there is a large body of literature about the biology of this insect and its interactions with host plants, going back more than 100 years. More recently, several research groups have initiated projects to study plant defense against aphids using Arabidopsis and the green peach aphid as a model system (de Vos et al., 2007; Louis and Shah, 2013). TuMV is a positive-strand RNA virus that infects not only Arabidopsis but also hundreds of other species in more than 40 plant families (Walsh and Jenner, 2002). It is considered to be one of the most damaging viruses for vegetable crops worldwide (Tomlinson, 1987; Nguyen et al., 2013; Yasaka et al., 2015) and is transmitted by the green peach aphid and many other aphid species in both natural and agricultural settings (Shattuck, 1992). Largely due to its ability to systemically infect Arabidopsis (Sánchez et al., 1998; Martín Martín et al., 1999), TuMV has become a model for potyvirus-host interactions (Walsh and Jenner, 2002).In this study, we investigate the role of phytohormone signals in TuMV’s ability to suppress plant defense and enhance aphid fecundity during infection of host plants. First, we show that TuMV infection induces SA and ET accumulation in Arabidopsis. Next, using genetic and pharmacological analyses, we demonstrate that ET signaling is necessary for TuMV-initiated suppression of plant defense responses and enhanced aphid reproduction in plants. Further, we show that expression of the viral protein Nuclear Inclusion a-Protease (NIa-Pro) alters ET responses and that ET is also required for NIa-Pro’s role in suppressing aphid-induced defense in virus-infected plants. This molecular, biochemical, and genetic evidence reveals that TuMV may modulate ET responses not only to increase plant susceptibility to infection but also to increase vector performance.