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.