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.