Discussion
The beneficial effects of microbial facultative endosymbionts on aphid hosts are well recognized, especially when aphids are challenged by biotic and abiotic stresses. On the other hand, these endosymbionts usually impair aphid growth and development, or suppress reproduction under normal environment as physiological costs (Chen, Montllor, & Purcell, 2000; Ferrari, Darby, Daniell, Godfray, & Douglas, 2004; Oliver, Moran, & Hunter, 2005). Facultative endosymbionts in pea aphids including H. defensa , Spiroplasma , Rickettsiadecrease longevity, fecundity and/or body weight of the aphid (Fukatsu, Tsuchida, Nikoh, & Koga, 2001; Polin, Simon, & Outreman, 2014; Russell, & Moran, 2006). By contrast, Serratia -infected aphids developed more rapidly than Serratia -free aphids on M. truncatula . Similarly, S. symbiotica accelerated cedar aphidCinara cedri growth as well by directly providing essential amino acids to host, gradually turning into an obligate endosymbiont (Lamelas et al., 2011). However, few amino acid metabolic genes have been annotated in the genome of S. symbiotica in pea aphids to supply nutrition (Manzanomarin, Lamelas, Moya, & Latorre, 2012). Since the field population of pea aphids had a relatively high infectious rate ofS. symbiotica , we speculated that S. symbiotica was likely involved in the early aphid feeding and colonization on Medicagoplants by supplying sufficient nutrition leading to accelerated population growth (Figure S1). In the current study, we demonstrated that S. symbiotica sharply up-regulated ApHRC in salivary glands of pea aphids, which efficiently suppressed Ca2+ signal and plant defense.
S. symbiotica is usually localized freely within hemolymph or near the bacteriocytes within pea aphids (Moran, Russell, Koga, & Fukatsu, 2005). S. symbiotica infection could impair the cellular immunity of pea aphid and decreased indole-3-lactate, an antioxidant, which may down-regulate ROS in aphid hemolymph (Burke, Fiehn, & Moran, 2010). Also, genome sequencing revealed that the IMD pathway along with many immune genes such as those encoding peptidoglycan recognition proteins and AMPs are lost in the pea aphid, possibly facilitating association of aphids with microbial symbionts (Gerardo et al., 2010). Interestingly, S. symbiotica increased drastically the expression of ApHRC , a gene encoding a presumably secretory protein in salivary glands (Figure 3B). Although little is known concerning its function in aphid immune response to the infection of S. symbiotica , we suspected that up-regulation of ApHRC possibly suppressed immune responses of aphid during the infection of S. symbiotica via quenching the early elevation of the Ca2+ signal as well.
Wounding caused by aphid feeding elicits plant Ca2+influx, a signal that turns on down-stream defenses, such as sieve element occlusion and ROS accumulation to prevent aphid feeding (Sun, Voorrips, Kaauwen, Visser, & Vosman, 2020). However, aphids secret salivary effectors to maintain constant phloem feeding (Mutti et al., 2008). Notably, several effectors have calcium-binding domains, such as Armet, that suppresses plant Ca2+ signal to facilitate aphid feeding (Cui et al., 2019). Here, Serratia infection aphids, either infested by aphids or infiltrated with saliva, led to weaker ROS and less H2O2 concentration in plant leaves, possibly attributing to the inhibition of Ca2+ signal via Ca2+-binding inM. truncatula (Tian et al., 2019). Consistent with this notion, silencing ApHRC significantly decreased the phloem ingestion time of aphids and induced higher ROS in M. truncatula , suggesting that ApHRC was a Serratia -induced salivary effector that improved aphid feeding. In addition to ROS, JA and SA signaling pathways have been widely reported to be involved in plant resistance against aphids (Mohase & Der Westhuizen, 2002; Selig, Keough, Nalam, & Nachappa, 2016; Sun et al., 2020). Since silencing ApHRC in the salivary glands or expression of ApHRC in plants only affectedLOX2 and PAL but was not significant for AOS2 ,NPR1 and PR1 , we conclude that JA and SA signaling pathways are only weakly affected by ApHRC. By contrast, the infestation of Serratia -infected aphids triggered weaker JA and SA signaling pathways than Serratia -free aphid in terms of all measured marker genes, indicating that some other salivary effectors ofSerratia -infected aphids down-regulated the JA and SA signaling pathways instead of ApHRC . Since a number of differentially expressed genes were affected by Serratia infection in our transcriptomic data of salivary glands, their function in suppression of plant defenses may be responsible for the discrepancy betweenSerratia -infection and ApHRC effects in activation of JA and SA signaling pathways. For example, Serratia -infected aphid could decrease the expression of a chemosensory protein (CSP) MP10 known to up-regulate JA and SA pathways of the host plant (Rodriguez, Stam, Warbroek, & Bos, 2014). Furthermore, HRC was also annotated in several other aphid species including Myzus persicae ,Diuraphis noxia , and Rhopalosiphum maidis , suggesting that the function of HRC might be conserved in aphids (Figure S3). In addition, a recent study showed that the mutation of a HRC in wheat enhanced the resistance against Fusarium head blight (Li et al., 2019). Most likely, HRC could down-regulate the Ca2+ responses to fungi infection, conferring susceptibility to the fungal diseases in wheat. Suppressing plant defense, as a newly discovered function of S. symbiotica , may explain its high prevalence in our field sampling compared with other facultative symbionts (Figure S1).
Growing evidence suggests that microbial mutualistic symbioses of insects could be orally secreted into a plant and directly manipulate the plant defenses (Chung, Rosa, Hoover, Luthe, & Felton, 2013). For example, a psyllid bacterial endosymbiont Candidatus Liberibacter psyllaurous could suppress JA and SA defensive signaling pathways of tomato plants (Casteel, Hansen, Walling, & Paine, 2012). The Colorado potato beetle secretes symbiotic bacteria to elicit SA-regulated defense while suppressing the efficient JA signaling pathway (Sorokan, Burkhanova, Benkovskaya, & Maksimov, 2019). Since most of the symbionts reside in bacteriocytes, gut, and hemolymph of insects, it is more common that the endosymbionts modulate the insect-plant interaction by indirectly regulating the transcripts of insect salivary gland genes rather than directly being secreted into the plant. We have shown that, on the basis of the transcriptome of salivary glands, the infection ofS. symbiotica could modify a number of salivary proteins of pea aphids (Table S5-S8). Our study has revealed a novel strategy employed by aphids where they host microbial facultative symbionts to benefit their own feeding. Serratia -infected aphids are thus more likely to be successful in host colonization and population expansion onMedicago plants.