Results
S. symbiotica Improves Aphid Feeding . To determine the effect of S. symbiotica on aphid feeding activity, we establishedSerratia -rebuilt aphids by injecting the hemolymph ofSerratia -infected aphids into Serratia -free aphids to exclude antibiotic influence. Electrical penetration graph (EPG) was used to monitor the feeding behavior of pea aphid on M. truncatula . Serratia -free aphids spent more time secreting saliva into sieve elements phase (E1 wave) and less time ingesting phloem (E2 wave) than Serratia -infected andSerratia -rebuilt aphids (Figure 1a and b). TheSerratia -rebuilt aphids began the first phloem ingestion phase significantly earlier than Serratia -free aphids although such difference was not statistically significant betweenSerratia -infected and Serratia -free aphids (Figure 1c). Shorter salivary secretion and longer passive feeding suggested that harboring S. symbiotica promoted feeding efficiency of pea aphids (Table S1).
Localization of S. symbiotica and B. aphidicola in Aphids. Fluorescence in situ hybridization (FISH) was used to determine the locations of S. symbiotica and B. aphidicolain pea aphids. As expected, only B. aphidicola was detected in bacteriocytes of Serratia -free aphids (Figure 1d), WhereasS. symbiotica was also seen near bacteriocytes inSerratia -infected and rebuilt aphids (Figure 1e and f).
Serratia-infected Aphids Avoided Triggering Strong Plant Defense. To determine the effect of Serratia -infection on aphid feeding-induced plant defense, we analyzed defense gene expression inM. truncatula infested by Serratia -free vs.Serratia -infected aphids. Compared with Serratia -free aphids, Serratia -infected aphids triggered lower gene expression levels of PAL, NPR1 , PR1 in the SA pathway, as well asAOS2 and LOX2 in the JA pathway (Figure 2a-e). Consistently, the EPG experiment indicated that Serratia -infected aphids had longer feeding duration than Serratia -free aphids.
We then assessed the ROS levels in plants after aphid infestation or saliva infiltration. Upon 6h infestation, Serratia -infected aphids triggered less H2O2 accumulation in plant leaves than Serratia -free aphids (Figure 2f). Furthermore, infiltration of saliva from Serratia -free aphids for 6 h led to a stronger fluorescence than from Serratia -infected aphid as well as mock-infiltration (Figure 2g). Presumably,Serratia -infected aphids would suffer less from ROS defense thanSerratia -free aphids, in agreement with the observation thatSerratia -infected aphids spent less time probing relative toSerratia -free aphids
Since Ca2+ is a ubiquitous signal that activates plant defense, we further studied the effect of aphid saliva on the cytosolic Ca2+ dynamics in plants using CaMV35S: GCaMP3 transgenic N. benthamiana . Saliva collected fromSerratia -free and Serratia -infected aphids both induced a robust Ca2+ signal within the initial 90 s in a 300 s time course whereas 15% sucrose control did not (Figure 2h). After 90 s however, Ca2+ signal decreased substantially in plants infiltrated with saliva from Serratia -infected aphids but remained high in plants infiltrated with saliva fromSerratia -free aphids for the remaining time period (Figure 2I; Movie S1-S3). Apparently, saliva of Serratia -infected aphids significantly suppressed Ca2+ signal.
S. symbiotica Modulates Gene Expression in Aphid Salivary Glands. To assess the effect of S. symbiotica infection on the gene transcription of salivary glands of pea aphids, the transcriptomic analyses were conducted using salivary glands of Serratia -free and Serratia -infected aphids. Of the 18,598 annotated genes,S. symbiotica significantly down-regulated the expression of 373 genes and up-regulated 347 genes (Figure 3a; Table S7, S8). Among the differentially expressed genes, 17 up-regulated and 37 down-regulated genes were predicted to contain signal peptides (Table S5 and S6). A gene annotated with ApHRC was almost 10 folds higher in salivary glands of Serratia -infected aphids than that ofSerratia -free aphids (Figure 3b). The 2.3kbp ApHRC encodes a 448 amino acid protein with a predicted signal peptide and two putative Ca2+-binding domains (Figure S2).
ApHRC expressed preferentially in a symmetrically disposed pairs of large secretory cells of the middle-lower cells of principal salivary glands (Figure 3c). Increase expression in the salivary glands and in the aphid body was detected in the presence of S. symbiotica(Figure 3d and e).
ApHRC Facilitated Feeding of Serratia-infected Aphids. To investigate whether ApHRC influenced the feeding ofSerratia -infected aphid, RNAi was performed to silenceApHRC. Twenty-four h after dsHRC -RNA injection, theApHRC expression level decreased 53% ± 12% in the body and 95% ± 4% in salivary glands (Figure 4a and b). ApHRC -silencedSerratia -infected aphids displayed prolonged salivary secretion (E1 wave), decreased the phloem ingestion phase (E2 wave) (Figure 4c and d), and increased intracellular punctures (pd wave, i.e. more navigation time to the phloem) (Figure 4e), which inevitably led to reduced feeding efficiency (Table S2).
ApHRC Suppresses Plant Defense by Eliminating the Ca2+ Elevation. Silencing ApHRC inSerratia -infected aphids resulted in higher PAL and lowerLOX2 expression, but no change in PR1 , NPR1 andAOS2 (Figure 4f-j). Furthermore, after 6 h infestation,ApHRC- silenced aphid produced significantly more H2O2 and stronger green fluorescence than the dsGFP control aphids (Figure 4k and l), suggesting that ApHRC can efficiently suppress plant ROS signals. The Ca2+ dynamics resembled that of Serratia -free aphids (Figure 4m and n; Movie S4-S6). These results indicatedApHRC was able to suppress the plant Ca2+signal during aphid infestation.
Expression of ApHRC in Plants Facilitated Feeding ofApHRC-silenced Serratia-infected Aphids . To further determine the effects of overexpression of ApHRC on plant defense and the feeding activity of ApHRC- silencedSerratia -infected aphids, we transiently expressed the full-length of ApHRC in M. truncatula  by agroinfiltration. Overexpression of ApHRC in M. truncatula  shortened salivary secretion (E1 wave) and elongated the duration of phloem feeding (E2 wave) (Figure 5a and b; Table S3).  Induced PAL and suppressed LOX2 expression was also observed (Figure 5c-g). While H2O2 was abundant in the plant cells in the vector-infiltrated plants, the ApHRC infiltrated plants barely accumulated any (Figure 5h and i). Therefore, the transient expression of ApHRC in M. truncatula leaves led to the inhibition of plant defense responses.
The Infection of S. symbiotica Improves the development of Pea Aphids . To determine the effect of S. symbioticainfection on the performance of pea aphids, offspring number, mean relative growth rate (MRGR) and the developmental duration of nymphs were compared between Serratia -free aphids andSerratia -infected aphids when reared onM. truncatula .S. symbiotica infection did not significantly affect MRGR and offspring number of pea aphids (Figure 6a and b). By contrast,Serratia -infected aphids had significantly shorter 2nd instar and 3rd instar duration than those of Serratia -free aphids (Figure 6c). Therefore,S. symbiotica infection improved aphid development.