1 INTRODUCTION
Plant hosts have defenses to counter attacks from antagonists such as
herbivores and pathogens (Pandey, Ramegowda, & Senthil-Kumar, 2015;
Miller, Costa Alves, & Van Sluys, 2017). For example, the jasmonic acid
pathway often regulates plant defenses against herbivores, while the
salicylic acid pathway often regulates defenses against pathogens
(Koornneef & Pieterse, 2008; Thaler, Humphrey, & Whiteman, 2012).
While many studies have assessed plant responses to particular
antagonists, plants are often challenged by many stressors concurrently,
and plant defenses can depend on the order in which antagonists arrive
on plants (Thaler et al., 2012; Nejat & Mantri, 2017). For example,
herbivores often limit plant defenses against pathogens when they arrive
first on plants, but herbivores often have few impacts on plant defenses
against pathogens when they arrive after pathogens on plants (Okada,
Abe, & Arimura, 2015; Lin et al. , 2019). In other contexts,
certain organisms ‘prime’ pathways, promoting defense against subsequent
organisms activating the same pathway, such that attack order may not
matter (Mauch-Mani, Baccelli, Luna, & Flors, 2017; Ramírez-Carrasco,
Martínez-Aguilar, & Alvarez-Venegas, 2017).
Biotic stressors may also alter the nutritional quality of plants by
regulating amino acid metabolism (Casteel et al., 2014; Zhou, Lou, Tzin,
& Jander, 2015), which can alter the feeding behavior and nutrient
uptake by subsequent herbivores (Behmer, 2009; Zhu, Poelman, & Dicke,
2014). For example, the composition of free amino acids constitutively
changes in leaves of soybean plants in response to soybean aphids
(Chiozza, O’Neal, & MacIntosh, 2010). Tomato yellow leaf curl
virus also alters the nutritional quality of tomato plants by affecting
free amino acid levels in phloem, which alters the amino acid
composition of whitefly (Bemisia tabaci ) honeydew (Guo et al.,
2019). However, few studies have explored how the diversity and identity
of attacking organisms, and variation in the order of attack, affect
nutritional traits of plants.
While there has been considerable research on the jasmonic and salicylic
acid pathways, to understand complexities of plant defense it is
necessary to assess how biotic antagonists mediate other signaling
pathways (e.g., Lacerda, Vasconcelos, Pelegrini, & Grossi de Sa, 2014;
Suzuki, 2016). Moreover, it is key to assess how changes in plant
defense correlate with plant nutrients. For example, plants in
low-nitrogen soil often adopt carbon-based defenses, while plants grown
with fertilizer often accumulate more nitrogenous toxins (Cipollini,
Walters, & Voelckel, 2017). Nitrogen in plants may also affect both
pathogens and herbivores through synthesis of defensive metabolite,
nitric oxide, and by nitrogen mobilization (War et al., 2012; Mur,
Simpson, Kumari, Gupta, & Gupta, 2017). However, few studies have
correlated effects of multiple biotic stressors on both plant chemical
signaling and nutritional properties (Petek et al., 2014; Su et al.,
2016).
We addressed these knowledge gaps by assessing the response ofPisum sativum plants to attack from a piercing-sucking vector
herbivore, the pea aphid (Acrythosiphon pisum ), a chewing
non-vector herbivore, the pea leaf weevil (Sitona lineatus ), and
an aphid-borne pathogen, Pea-enation mosaic virus (PEMV). These
organisms co-occur in ecosystems of eastern Washington and northern
Idaho, USA, and interactions between them can affect plant traits and
signaling pathways affecting insects and pathogens (Chisholm,
Eigenbrode, Clark, Basu, & Crowder, 2019; Bera, Blundell, Liang,
Crowder, & Casteel, 2020). However, the order in which herbivores and
pathogens arrive on hosts, which varies across sites (Chisholm et al.,
2019), may impact plant traits and defenses. To address this, we varied
the diversity, identity, and order of attack among this community of
biotic antagonists and assessed resulting changes in gene expression and
phytohormones related to plant defense and nutrition. Our study revealed
how plant responses to diverse stressors can mediate complex species
interactions within a pathosystem.