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Article

Pea Aphid (Acyrthosiphon pisum) Host Races Reduce Heat-Induced Forisome Dispersion in Vicia faba and Trifolium pratense

by
Maria K. Paulmann
1,2,
Linus Wegner
2,3,
Jonathan Gershenzon
1,
Alexandra C. U. Furch
2,* and
Grit Kunert
1,*
1
Max Planck Institute for Chemical Ecology, Department of Biochemistry, Hans-Knöll-Str. 8, D-07745 Jena, Germany
2
Plant Physiology, Matthias Schleiden Institute for Genetics, Bioinformatics and Molecular Botany, Faculty of Biological Science, Friedrich Schiller University Jena, Dornburger Straße 159, D-07743 Jena, Germany
3
Institute of Botany, Justus Liebig University, Heinrich-Buff-Ring 38, 35292 Giessen, Germany
*
Authors to whom correspondence should be addressed.
Plants 2023, 12(9), 1888; https://doi.org/10.3390/plants12091888
Submission received: 7 February 2023 / Revised: 21 April 2023 / Accepted: 28 April 2023 / Published: 6 May 2023
(This article belongs to the Special Issue Plant-Aphid Interactions: From Genes to Ecosystems)
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Abstract

:
Although phloem-feeding insects such as aphids can cause significant damage to plants, relatively little is known about early plant defenses against these insects. As a first line of defense, legumes can stop the phloem mass flow through a conformational change in phloem proteins known as forisomes in response to Ca2+ influx. However, specialized phloem-feeding insects might be able to suppress the conformational change of forisomes and thereby prevent sieve element occlusion. To investigate this possibility, we triggered forisome dispersion through application of a local heat stimulus to the leaf tips of pea (Pisum sativum), clover (Trifolium pratense) and broad bean (Vicia faba) plants infested with different pea aphid (Acyrthosiphon pisum) host races and monitored forisome responses. Pea aphids were able to suppress forisome dispersion, but this depended on the infesting aphid host race, the plant species, and the age of the plant. Differences in the ability of aphids to suppress forisome dispersion may be explained by differences in the composition and quantity of the aphid saliva injected into the plant. Various mechanisms of how pea aphids might suppress forisome dispersion are discussed.

1. Introduction

Translocation of photoassimilates, nitrogen-rich compounds and signaling molecules in vascular plants is carried out by the transport conduits of the phloem—the sieve elements (SEs). However, SEs are targeted by aphids and other piercing–sucking insects that feed on phloem sap [1,2]. Plants have developed two main mechanisms to prevent the loss of phloem sap in response to mechanical damage [3] or phloem-feeding insects [4,5,6]. (1) Long-term occlusion of SEs is achieved by the calcium-ion (Ca2+)-triggered accumulation of callose at the sieve pores and the plasmodesmata, while (2) short-term occlusion is achieved by phloem proteins (P-proteins) [3,7,8,9,10,11]. Among the occluding P-proteins are the spindle-like forisomes, which are unique to legumes [11,12]. In response to damage and subsequent Ca2+ influx into the SE, forisomes expand reversibly until they are fully dispersed and clog the SE [10,13,14]. Based on this behavior, forisomes can be considered as natural Ca2+ indicators [15]. Forisomes have been shown to stop mass flow in vitro [16] and are believed to have a similar function in planta [11,17]. The expansion of forisomes occurs at a much faster rate than callose deposition, and so reduces phloem sap loss until callose deposition is complete [3].
Aphids typically cause little damage to plant tissue because they use their stylets to navigate in between plant cells [18,19,20]. During this process, aphids inject saliva into the plant [21,22]. One type of saliva, called gelling saliva, is secreted into the apoplastic space where it gelatinizes, and the aphid subsequently pushes its stylet through the gel so that the gel forms a sheath [23]. Besides protecting the stylet from mechanical damage and possibly manipulating plant responses [21], the gelling saliva has been suggested to seal cell wall sites where non-SE cells have been pierced [19,24]. This could, for example, prevent influx of Ca2+ ions and subsequent aphid recognition by the plant. Elicitors present in the aphid saliva can also lead to plant recognition of the aphid’s presence [25]. During navigation through the plant apoplast, the aphid occasionally pierces plant cells to orient itself [19,26] and secretes a second, watery type of saliva into the cell [27]. Elicitors such as Mp10 may then be recognized by the plant [28], which launches plant defense responses that reduce aphid fecundity [28]. Induced defenses, such as callose and forisomes, can be located in the phloem [29,30,31,32] leading to SE or stylet clogging, which impairs the uptake of sap [5,33] and consequently aphid performance.
Plant defense responses to phloem feeding can in turn be manipulated by effectors in the aphid saliva [26,34,35]. These effectors may prevent hypersensitive cell death or suppress other defenses, such as Ca2+-triggered occlusion via callose or forisomes [36,37,38,39]. For example, forisome dispersion is reversible in vitro through application of the saliva of the aphid Megoura viciae [Buckton] [38]. Inter- and intraspecific variation of aphid saliva composition [37,40,41] could therefore be responsible for preventing phloem defenses such as occlusion due to forisomes. Saliva composition differences could be involved in the ability of the specialist aphid Acyrthosiphon pisum [Harris] to feed readily on Vicia faba [L.] and not induce forisome dispersion while the generalist aphid Myzus persicae [Sulzer] is not able to establish feeding on V. faba, a plant on which it triggers forisome dispersion [4,42].
The specialist A. pisum is a species complex of at least 15 genetically different host races. Each host race is native to one or a very few closely related legume species and can survive and reproduce successfully only on its respective host plant [43,44]. However, all pea aphid host races are able to feed on V. faba, the so-called universal host plant. Plant responses to pea aphid feeding such as changes in phytohormones [45] and metabolites [46] have been shown to depend on the pea aphid host race. These pea aphid host race-specific responses may be triggered through differences in aphid saliva composition [47]. Feeding behavior of different pea aphid host races indicated that the phloem might be involved in this pea aphid—host plant specificity [31]. Thus, forisomes located in SEs probably play a role in this aphid—host plant specificity. Hence, we hypothesize that the pea aphid host races are able to suppress forisome dispersion on their native host plant and are thus able to feed, but are not able to suppress, forisome dispersion on nonhost plants.
Since forisome dispersion induced by individual aphids is very difficult to detect and investigate in an experimental context [4,42,48], we triggered dispersion with a heat stimulus and then examined whether pea aphid host races were able to prevent forisome dispersion on native host and nonhost plants as well as on the universal host plant. We found that pea aphid host races were able to suppress heat-triggered forisome dispersion. This however, was dependent on the specific plant species—pea aphid host race combination and on plant age. To see whether the pea aphid host races used for this investigation differed in their saliva composition, watery saliva of different pea aphid host races was collected, and salivary proteins were compared. We found that aphid saliva composition differed between the pea aphid host races and might therefore indeed be responsible for the host race-dependent forisome reaction.

2. Results

2.1. Suppression of Forisome Dispersion Depends on Host Plant—Aphid Host Race Combination as Well as on Plant Age

The ability of different A. pisum host races to suppress forisome dispersion in different host plants was studied through application of a heat stimulus to the leaf tip while monitoring forisome responses in the same leaf. Heat stimulation is a strong trigger for forisome dispersion [3]. We assessed the ability of aphids to reduce forisome dispersion after 48 h of aphid infestation.
For all investigated legume species, the distance of the forisome to the heat-stimulus site did not influence the ratio of dispersed to all forisomes (Table 1).
Table
Table 1. Statistical values for the analyses of the forisome dispersion ratios of different legume species in response to heat stimulation with variation in the distance of the observed forisome to the site of heat stimulation, variation in plant age, and variation in the host race of the infesting pea aphid (Acyrthosiphon pisum; treatment).
Table 1. Statistical values for the analyses of the forisome dispersion ratios of different legume species in response to heat stimulation with variation in the distance of the observed forisome to the site of heat stimulation, variation in plant age, and variation in the host race of the infesting pea aphid (Acyrthosiphon pisum; treatment).
PlantExplanatory VariableDeviancep-Value
Vicia faba (Figure 1)
distance−0.7770.378
plant age−0.3020.582
treatment−2.1910.534
treatment: plant age−7.8580.049
distance: plant age−0.0010.970
distance: treatment−0.8570.836
distance: treatment: plant age−0.7770.396
Trifolium pratense (Figure 2)
distance−0.0060.939
plant age−4.6430.031
treatment−9.5890.022
treatment: plant age−2.2840.516
distance: plant age−0.0630.802
distance: treatment−3.4860.323
distance: treatment: plant age−0.4530.929
Pisum sativum (Figure 3)
distance−0.6460.422
treatment−2.7200.437
distance: treatment−1.8800.598
Significant p-values are highlighted in bold.
Plants 12 01888 g001 550
Figure 1. Influence of different A. pisum host races on the dispersion behavior of forisomes in the universal host plant V. faba. (A) V. faba forisome in its condensed (left panel, dashed line) and its dispersed (right panel, putative location indicated by asterisk) state. Depicted are the ratios of fully dispersed forisomes to all observed forisomes after heat stimulation (B) in various host race treatments on plants at both ages, (C) in various plant ages (indicated by fill shade), and (D) in various aphid host race treatments. Different lower-case letters above bars indicate significant differences (p ≤ 0.05). Data were analyzed by a Bernoulli GLM. Further statistical information can be found in Table 1. Numbers in the bars indicate the total number of forisomes observed for the respective treatment (N). MR—Medicago host race (green); PR—Pisum host race (orange); TR—Trifolium host race (blue).
Figure 1. Influence of different A. pisum host races on the dispersion behavior of forisomes in the universal host plant V. faba. (A) V. faba forisome in its condensed (left panel, dashed line) and its dispersed (right panel, putative location indicated by asterisk) state. Depicted are the ratios of fully dispersed forisomes to all observed forisomes after heat stimulation (B) in various host race treatments on plants at both ages, (C) in various plant ages (indicated by fill shade), and (D) in various aphid host race treatments. Different lower-case letters above bars indicate significant differences (p ≤ 0.05). Data were analyzed by a Bernoulli GLM. Further statistical information can be found in Table 1. Numbers in the bars indicate the total number of forisomes observed for the respective treatment (N). MR—Medicago host race (green); PR—Pisum host race (orange); TR—Trifolium host race (blue).
Plants 12 01888 g001
Plants 12 01888 g002 550
Figure 2. Influence of different A. pisum host races on the dispersion behavior of forisomes in the native host plant T. pratense. (A) T. pratense forisome in its condensed (left panel, dashed line) and its dispersed (right panel, putative location indicated by asterisk) state. Depicted are the ratios of fully dispersed forisomes to all observed forisomes after heat stimulation (B) in various host race treatments on plants at both ages, (C) in various plant ages (indicated by fill shade), and (D) in various aphid host race treatments. Different lower-case letters above bars indicate significant differences (p ≤ 0.05). Data were analyzed by a Bernoulli GLM. Further statistical information can be found in Table 1. Numbers in the bars indicate the total number of forisomes observed for the respective treatment (N). MR—Medicago host race (green); PR—Pisum host race (orange); TR—Trifolium host race (blue).
Figure 2. Influence of different A. pisum host races on the dispersion behavior of forisomes in the native host plant T. pratense. (A) T. pratense forisome in its condensed (left panel, dashed line) and its dispersed (right panel, putative location indicated by asterisk) state. Depicted are the ratios of fully dispersed forisomes to all observed forisomes after heat stimulation (B) in various host race treatments on plants at both ages, (C) in various plant ages (indicated by fill shade), and (D) in various aphid host race treatments. Different lower-case letters above bars indicate significant differences (p ≤ 0.05). Data were analyzed by a Bernoulli GLM. Further statistical information can be found in Table 1. Numbers in the bars indicate the total number of forisomes observed for the respective treatment (N). MR—Medicago host race (green); PR—Pisum host race (orange); TR—Trifolium host race (blue).
Plants 12 01888 g002
Plants 12 01888 g003 550
Figure 3. Influence of different A. pisum host races on the dispersion behavior of forisomes in the native host plant P. sativum. (A) P. sativum forisome in its condensed (left panel, dashed line) and its dispersed (right panel, putative location indicated by asterisk) state. (B) Depicted are the ratios of fully dispersed forisomes to all observed forisomes after heat stimulation in various aphid host race treatments. No significant differences were observed. Data were analyzed by a Bernoulli GLM. Further statistical information can be found in Table 1. Numbers in the bars indicate the total number of forisomes observed for the respective treatment (N). MR—Medicago host race (green); PR—Pisum host race (orange); TR—Trifolium host race (blue).
Figure 3. Influence of different A. pisum host races on the dispersion behavior of forisomes in the native host plant P. sativum. (A) P. sativum forisome in its condensed (left panel, dashed line) and its dispersed (right panel, putative location indicated by asterisk) state. (B) Depicted are the ratios of fully dispersed forisomes to all observed forisomes after heat stimulation in various aphid host race treatments. No significant differences were observed. Data were analyzed by a Bernoulli GLM. Further statistical information can be found in Table 1. Numbers in the bars indicate the total number of forisomes observed for the respective treatment (N). MR—Medicago host race (green); PR—Pisum host race (orange); TR—Trifolium host race (blue).
Plants 12 01888 g003
Forisome dispersion in V. faba was dependent on the pea aphid host race—plant age combination (p = 0.049; Figure 1B; Table 1). Medicago host race (MR) and Pisum host race (PR) aphids suppressed forisome dispersion on three-week-old V. faba compared to dispersion on uninfested control plants. On four-week-old plants, however, the dispersion rate was significantly higher in MR and PR infested plants compared to control plants. Trifolium host race aphids (TR) on V. faba did not alter forisome dispersion compared to the uninfested control, but dispersion was reduced on four-week-old plants compared to three-week-old plants.
Forisome dispersion in T. pratense [L.] was influenced by plant age (p = 0.031; Figure 2C) and the host race of the infesting aphid (p = 0.022; Figure 2D; Table 1) independently from each other (p = 0.516; Table 1). With increasing plant age, forisomes became less responsive to heat stimulation. Among host races, the non-native PR and particularly the native TR were able to suppress forisome dispersion in a significant manner compared to uninfested control plants.
On P. sativum [L.], pea aphids could not suppress forisome dispersion irrespective of whether native or non-native host races were used (p = 0.437; Figure 3B; Table 1).
The results for the three different plant species show that forisome responses depend significantly on the host race of the infesting aphid and plant age, as well as on the plant species.

2.2. Aphid Saliva Composition Depends on the Pea Aphid Host Race

The differential impact of the various aphid host races on forisome dispersion might be explained by differences in aphid saliva composition. In order to investigate this, we collected aphid watery saliva and analyzed it by gel electrophoresis. Visualization of native proteins revealed that PR was lacking a protein band at an apparent molecular weight of around 200 kDa (Figure 4A, arrowhead) that was present in the saliva of MR and TR. Further, we observed protein bands at an apparent molecular weight larger than 250 kDa for PR and TR, but not for MR (Figure 4A, asterisk).
When denatured samples from the same collections were analyzed by SDS-PAGE, the bands at molecular weights larger than 250 kDa in the PR and TR lanes could no longer be observed (Figure 4B), which indicates that these protein bands in the native protein gel likely arose from multimeric complexes. Additionally, the saliva of PR showed a distinct difference to that of MR and TR in that only a single band could be observed at 100 to 110 kDa instead of a double band (Figure 4B, arrowhead).
Carolan and colleagues previously investigated pea aphid saliva composition through SDS-PAGE and proteomics [39,49]. We correlated the proteins they reported with those in our gels based on their apparent molecular weight (compare Figure 4; Table 2). Previously reported proteins that appear to be present in our samples include a metalloprotease (130–150 kDa), a calcium-binding protein (regucalcin, 41 kDa), an angiotensin-converting enzyme (ACE, 100–115 kDa) and a putative sheath protein (130–150 kDa) [39,49]. Not all proteins could be found in all samples, such as one homologue of the ACE, which was only present in MR and TR saliva (Figure 4B, arrowhead, 100 kDa).

3. Discussion

In order to see whether pea aphid host races are able to suppress forisome dispersion in Fabaceae plants and therefore, SE clogging, we utilized heat stimulation by burning to trigger a strong Ca2+ influx into the SE [3,50]. An increase in Ca2+ concentration is the only reason for forisome dispersion [14]. If aphids are able to suppress forisome dispersion they consequently must be able to manipulate Ca2+ levels in SEs. Additionally, if pea aphid host races are able to suppress forisome dispersion in spite of such a strong trigger like heat stimulation, we hypothesize that they would also be able to suppress feeding-triggered Ca2+ influx and forisome dispersion.
Our results clearly show that suppression of heat-triggered forisome dispersion is not a general phenomenon in all interactions between legume host plants and their native pea aphid host races. Forisomes of different plant species reacted differently ranging from no alteration of forisome dispersion by aphid infestation as in P. sativum to obvious suppression of forisome dispersion by aphids as in T. pratense, especially but not solely by the native pea aphid host race. Whether forisome dispersion in V. faba was suppressed by aphids was dependent on the aphid host race and the age of the plant. These diverse dispersion patterns may be explained in different manners.
In order to fully disperse, forisomes have to come into contact with a certain amount of Ca2+ ions (Scheme 1) [50]. This is the only stimulus which leads to forisome dispersion [14]. Thus, forisomes can be considered as Ca2+ sensors, and non- or only partially dispersed forisomes indicate insufficient amounts of Ca2+ in the SEs to trigger forisome dispersion [14,50].

3.1. Influence of the Damage Caused by Different Pea Aphid Host Races

Even though aphids cause little damage to the plant since they navigate their stylet through the apoplast, they regularly pierce cells to orient themselves and find the SEs [18]. This slight damage can still lead to local Ca2+ bursts [51], which may ultimately trigger forisome dispersion [4]. The higher the number of aphids penetrating a plant, or the higher the number of piercing events by one aphid, the greater the damage, and the more Ca2+ bursts should occur in plant cells. Following this logic, aphid-infested plants experience more damage and, consequently, more Ca2+ bursts than aphid-free control plants. In theory, this should lead to a higher proportion of dispersed forisomes. This was, however, not the case in our study (Figure 1, Figure 2 and Figure 3). On the contrary, especially in T. pratense, fewer forisomes dispersed after infestation with the native TR aphid (40.9%) compared to the control plants (80%, Figure 2). There was also the tendency that T. pratense infested with the native TR aphid showed less forisome dispersion (40.9%) than when infested with the non-native MR aphid (63.2%, Figure 2). If damage would have been the main driver in this interaction, native TR aphids would have caused less damage than MR aphids. However, the contrary is the case [31]. Previous experiments have shown that more aphids pierce SEs during a compatible plant—aphid interaction than during an incompatible interaction. Also, the time aphids of a compatible interaction spend during a compatible interaction navigating their stylets through the epidermis and mesophyll is longer than the time aphids spent during an incompatible interaction [31]. Thus, all in all, the observed forisome dispersion patterns cannot be explained by different degrees of damage caused by pea aphid host races, but must be attributed to host race-specific differences in physiology or behavior.

3.2. Possible Mechanisms of Forisome Dispersion Manipulation

The one and only way that forisomes change their conformation is via an increase of the Ca2+ concentration in the SEs [10,52]. Thus, since some aphid host races are able to suppress forisome dispersion, they must be able to suppress heat-induced Ca2+ level increases. There are several potential and not mutually exclusive ways of how Ca2+ levels in SEs and therefore forisome dispersion can be influenced by aphids: 1. By manipulation of signal transduction and consequently altered Ca2+ influx into the SE. 2. By manipulation of Ca2+ channels responsible for Ca2+ influx and efflux. 3. By differences in Ca2+ levels among plant species. 4. Through Ca2+-scavenging capacity of the aphid saliva. 5. By the quality and quantity of aphid saliva injected into the plant.

3.2.1. Signal Transduction and Forisome Dispersion

The Ca2+ influx into the cytosol of SEs that triggers forisome dispersion occurs through Ca2+ channels [53]. The Ca2+ channel opening can be mediated by electrophysiological reactions (elRs) [54,55]. Strong elRs along the plant vasculature can be triggered by heat stimulation (burning) of the leaf [3,14,50]. We have previously shown that forisome dispersion is triggered by variation potentials (VPs) and electropotential waves (EPWs), which are a combination of VP and action potential (AP) [55]. Since APs alone did not trigger forisome dispersion, VPs must be responsible for forisome dispersion. VPs, however, result from chemical components [56] or the propagation of pressure waves within the xylem vessels, which lead to local activation of mechano-sensitive Ca2+ channels of the SE plasma membrane [57,58,59]. Since the xylem is seldom pierced by nonmigrating parthenogenetic pea aphids [31], direct interference with the signal transduction along the xylem is quite unlikely. However, the decline in forisome dispersion with increasing plant age in T. pratense (from 68.1% to 43.7%, see Figure 2C) and aphid-free V. faba (from 73.7% to 55.6%, see Figure 1B) might result from changes in signal transduction. With increasing age, more lignin is deposited in the cell walls, increasing the rigidity of the vascular system [60]. This may negatively affect the hydraulic pressure wave necessary to transmit the VP and, hence, prevent VP-triggered forisome dispersion [61,62].

3.2.2. Manipulation of Ca2+ Channels Responsible for the Influx and Efflux of Ca2+

Upon arrival of a given signal, ion channel opening is needed to transfer Ca2+ from the apoplast or internal storage, such as the endoplasmic reticulum, into the cytosol. Different Ca2+ channels are known so far in plants [53,63,64], for example GLUTAMATE RECEPTOR-LIKE proteins (GLR) which are activated through amino acid ligands and necessary for VP propagation [65]. One of these channels (GLR3.3) is implicated in plant defense [66] and GLR3.4 is phloem-located [67]. Both could, therefore, be a target of aphid manipulation [6]. The inhibition of GLRs may be achieved through low molecular weight amino acid analogs present in the aphid watery saliva.
After stimulus-triggered Ca2+ increases, the cytosolic Ca2+ concentration has to be returned to the resting levels in order for forisomes to recondense [68]. This offers an opportunity for native aphids to manipulate forisome dispersion since an increased efflux rate would also negatively impact Ca2+-dependent forisome dispersion. The saliva of MR was shown to contain regucalcin (41 kDa, see Figure 4 and Table 2), which is a Ca2+ binding protein that is known to activate Ca2+ pumps [69]. Hence, MR may accelerate the Ca2+ efflux from the SE and reduce forisome dispersion as seen in three-week-old V. faba (from 73.7% to 56.2%, see Figure 1B). Whether such alteration of Ca2+ import/export channels really takes place and whether it can influence forisome dispersion still needs to be investigated.

3.2.3. Differences in Ca2+ Levels among Plant Species

Whereas the Ca2+ levels in SEs at resting conditions are at a universally low level of around 0.05 µM [50,70], Ca2+ levels differ between plant tissues and under different water potentials [71], and so might also differ between plant species in a way that affects the aphid’s ability to manipulate forisome dispersion. However, to date there is little information regarding the quantitative changes in Ca2+ levels in SEs [15,50]. It is known that forisomes of V. faba require 60–100 µM Ca2+ in order to disperse [50,72], and it is also known that Ca2+ concentrations required for forisome dispersion likely increase with forisome size [55]. Thus, since the forisomes of P. sativum are in general bigger than those of T. pratense [55], it is possible that the induced Ca2+ concentrations in SEs of P. sativum are higher than in those of T. pratense. Assuming that the saliva from some pea aphid host races has the Ca2+-scavenging capacity to suppress forisome dispersion in T. pratense (consistent with our results, forisome dispersion reduction PR 30%, TR 39.1%, Figure 2D), this capacity might not be enough to suppress the dispersion of the larger forisomes of P. sativum (consistent with no forisome dispersion reduction by aphids in our study, Figure 3B) with their potentially higher Ca2+ levels.

3.2.4. Ca2+-Scavenging Capacity of the Aphid Saliva

It has been discussed for some time that aphids might be able to suppress forisome dispersion by the Ca2+-scavenging capacity of their saliva [37,38,68,73]. Ca2+-scavenging proteins in aphid saliva have been reported for M. viciae [38], as well as A. pisum [39,74]. However, when feeding on P. sativum, the scavenging capacity of the investigated pea aphid saliva does not seem to be large enough, since none of the pea aphid host races in our study were able to suppress heat-induced forisome dispersion (see Figure 3).
In T. pratense, on the other hand, heat-triggered forisome dispersion is differentially affected by pea aphid host races (80% control plants, 63.1% MR, 50% PR, 40.9% TR, see Figure 2D), which could at least be partly explained by variation in the Ca2+-scavenging capacity of the aphid saliva. It is likely that the Ca2+-scavenging capacity differs between the different pea aphid host races, since, as discussed above, the forisomes of the investigated host plants vary in their sizes. Due to forisomes in P. sativum being larger than those in T. pratense [55], it can be assumed that more Ca2+ ions are needed for full dispersion. Consequently, it is likely that the saliva of PR aphids has a higher Ca2+-scavenging capacity than the saliva of TR aphids. This might explain why the non-native PR could substantially supress forisome dispersion on T. pratense from 80% to 50% (see Figure 2D).
That the Ca2+-scavenging capacity of saliva differs between pea aphid host races is further supported by the differences in protein composition presented (Figure 4) and previous research showing variation in pea aphid saliva composition among host races [38,39,75]. So far, only a few Ca2+-scavenging proteins (regucalcin, ARMET) are known [39,49,69,74] and the function of the vast majority of saliva proteins still needs to be elucidated. More Ca2+-scavenging proteins may be found in the future whose occurrence differs among the pea aphid host races.
Since V. faba is the universal host plant for all pea aphid host races, we hypothesized that all host races would be able to suppress heat-induced forisome dispersion on this plant. Yet, we only observed suppression for the MR and PR aphids and only in younger plants (from 73.7% to 56.2% for MR and to 47.6% for PR, Figure 1B). The ratio of dispersed forisomes in MR- and PR-infested older plants was even higher (+31.9% for MR, +6.9% for PR) than in aphid free plants of the respective age. Previous reports indicated that the MR was also not able to reverse forisome dispersion in preflowering V. faba plants [42,48]. Such a pattern indicates that the Ca2+-scavenging capacity of aphid saliva by itself is likely not sufficient to explain forisome dispersion ability, implicating a stronger role for Ca2+ channel manipulation as discussed above.

3.2.5. Quality and Quantity of Aphid Saliva

Most of the mechanisms for suppressing forisome dispersion discussed above can be reinforced if aphids inject more saliva into the plant. So far, we cannot quantify the amount of saliva injected by one aphid, but the amount of saliva injected into SEs depends also on the number of aphids feeding on a plant. Infestations by native host races result in more aphids reaching the phloem and feeding on the plant than infestations of non-native host races [31]. Since aphid feeding is associated with salivation into the plant [21,76], likely more saliva from native host races is injected into the host plant than from non-native host races. This assumption is supported by an aphid feeding study from Schwarzkopf and colleagues [31], which showed that nearly all TR aphids salivated into SEs on their native host plant T. pratense but only a low portion of non-adapted MR and PR aphids did so. This mechanism would nicely explain the strong suppression of forisome dispersion by the native TR on T. pratense from 80% to 40.9%) and the lower ability to supress forisome dispersion of the non-native MR (from 80% to 63.1%, see Figure 2D).
To further complicate matters, not only the amount of saliva secreted into a plant changes with plant species, but also the composition of the aphid saliva changes depending on the plant the aphid is feeding on [47,77]. This makes it even more difficult to pinpoint the precise mode of action involved in the suppression of forisome dispersion.

3.3. Closing Remarks

Since forisome-triggered SE occlusion depends on Ca2+ ions, Ca2+ scavenging and Ca2+ channel manipulation are two mechanisms that could act in concert to suppress forisome dispersion and thereby enable the different pea aphid host races to feed on their respective host plants. While Ca2+ scavenging prevents forisome dispersion as well as the opening of potential Ca2+-dependent channels in the SE, direct manipulation of the Ca2+ channels can prevent phloem defense responses even before the aphid pierces a given SE [4,42,64].
Regardless of the different mechanisms by which aphids might change forisome dispersion, we have to keep in mind that in this study we actually investigated the capacity of the aphids to suppress heat-induced forisome dispersion as a proxy for suppression of forisome dispersion induced by aphid feeding. Heat stimulation induces strong Ca2+ fluxes that may surpass feeding-triggered increases, and Ca2+ bursts and subsequent forisome dispersion might be much lower upon actual aphid infestation without heat treatment. Thus, if aphids are able to suppress forisome dispersion triggered by a heat stimulus, they might easily be able to suppress forisome dispersion triggered during feeding. Aphids that were not able to suppress heat-induced forisome dispersion in this study may be able to more readily cope with the lower Ca2+ concentrations in SEs that might occur during normal feeding. The fact that some host races are able to suppress heat-triggered forisome dispersion illustrates that aphids can counter this phloem defense mechanism and paves the way for future studies on the underlying mechanisms.

4. Materials and Methods

4.1. Plant and Aphid Cultivation

The experiments were conducted on Pisum sativum cultivar (cv) ‘Baccara’, Trifolium pratense cv ‘Dajana’ and Vicia faba cv ‘The Sutton’ since they represent either native host plants (P. sativum and T. pratense) or the universal host plant (V. faba) for pea aphid host races. This allowed us to investigate compatible and incompatible combinations of aphids and plants.
The plants were cultivated in 10 cm diameter plastic pots with a standardized soil mixture of Klasmann Tonsubstrat and Klasmann Kultursubstrat TS1 (proportion 7:20; Klasmann-Deilmann GmbH, Geeste, Germany). The temperature of the growth chamber was maintained between 20 and 22 °C and the relative humidity between 60 and 70%. Long day conditions (L16:D8) were used with an irradiance level of 100 to 150 µmol m−2 s−1 (Fluora lamps, Osram GmbH, Munich, Germany).
All A. pisum clones used were free of facultative endosymbionts and were previously obtained from ampicillin-treated naturally occurring clones. More information regarding these pea aphid clones can be found in [78,79]. The A. pisum Medicago host race (MR; clone ID218—obtained from L84), Pisum host race (PR; clone ID212—obtained from P123) and Trifolium host race (TR; clone ID210—obtained from YR2) were reared separately in small tents (Bugdorm; MegaView Science Co., Ltd., Taiwan) on V. faba under the plant growth conditions listed above.

4.2. Experimental Set-Up

Studies of local influences on forisome dispersion were executed on P. sativum (4 weeks), T. pratense (6–7 weeks) and V. faba (3–4 weeks) in their vegetative state before flowering. Aphid-free plants served as controls. Each plant species was infested separately with all three pea aphid host races. Whole plants were infested with around 150 to 200 apterous, parthenogenetic aphids of mixed age, which were allowed to feed for 48 h. To prevent the escape of aphids, all experimental plants were caged in tents according to aphid host race.
Plants were handled in a manner so as not to dislodge feeding aphids from the plants. For in vivo observations of forisome reactions, the phloem was exposed by removing the cortical cell layers from the lower side of the midvein with a razor blade as described previously [13,80]. The leaf was then fastened upside down onto an objective slide with double-sided adhesive tape. The exposed tissue was immersed in physiological bathing medium (2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 50 mM mannitol, 2.5 mM MES/NaOH buffer, pH 5.7) and allowed to incubate for one hour. The integrity of the tissue was subsequently analyzed with a light microscope (AXIO Imager.M2, Zeiss, Jena, Germany) using a 40 x water immersion objective (W-N Achroplan, Zeiss).
To trigger forisome dispersion, a heat stimulus was applied for 2 s with a match to the tip of the observation leaflet. For V. faba, this was leaf number 4, for P. sativum the second youngest, mature leaflet, and for T. pratense the central leaflet (compare Figure 5). In accordance with previous research [55], we limited the range of the distance from the stimulus to the observation site to 2 to 3 cm in V. faba, 1.5 to 3 cm in T. pratense and 1 to 2.5 cm in P. sativum. Each plant was used once and only plants with intact SEs and forisomes located on the downstream (basal) side of the SE were considered for experiments. It was recorded whether the forisomes dispersed, did not disperse or partially dispersed, which was counted as unsuccessful dispersion. Heat triggered forisome reactions were traced with a color camera (AXIOCAM 503 color, Zeiss) and micrographs processed with the ZEN (blue edition) software (Zeiss). The figures presented were compiled with Adobe Illustrator CS5 (Dublin, Ireland).

4.3. Aphid Saliva Collection and Preparation

On average, 100 aphids of all ages from each host race were used to collect saliva. For this, 1 mL sterile artificial diet (100 mM aspartic acid, 100 mM methionine, 100 mM serine, 15% sucrose, pH 7.2 (KOH) [38]) was plated between two Parafilm (Beemis Company Inc., Neenah, IW, USA) layers, which were covered by a cage to prevent the aphids from escaping. After 24 h, the aphids were removed and the remaining diet collected.
In order to concentrate saliva proteins larger than 3 kDa and cleanse the concentrate of surplus sugar and amino acids, the protocol of Will and colleagues [38] was complemented with multiple washing steps. The saliva-diet suspension of at least 6 collections was transferred to a Vivaspin 20 concentrator (3 kDa cutoff, PES; Sartorius, Göttingen, Germany) and the volume increased to 20 mL with 10 mM Tris-HCl (pH 7.3). After 1:20 h centrifugation at 4 °C and 4000× g, another 5 mL Tris-HCl were added and the centrifugation step was repeated. The remaining concentrate was again filled up with Tris-HCl to a final volume of 20 mL and spun for another 2 h. The supernatant was transferred to a Vivaspin 500 column (3 kDa cutoff, PES; Sartorius). After one hour of centrifugation at 4 °C and 4000× g, 500 µL Tris-HCl (pH 7.3) were added and the samples were centrifuged overnight. Afterwards 200 µL 10 mM Tris-HCl (pH 7.3) were added to the supernatant and the samples were centrifuged another five to six hours at 4000× g and one to two hours at 10,000× g. The retained saliva samples (ca. 15 to 20 µL each) were stored at −80 °C until further usage.

4.4. Determination of Protein Concentration and Saliva Compositions

The protein concentration of each concentrated saliva sample was determined by adding 2 µL to 50 µL Bradford solution (QuickStartTM Bradford Prot Assay; Bio-Rad Laboratories, Hercules, CA, USA) in a 96 well plate (PS, F-bottom, clear; Greiner bio-one, Frickenhausen, Germany). After 10 min incubation, the absorption was measured at 595 nm with a Tecan multi-well reader (infinite M200; Tecan Austria GmbH, Grödig/Salzburg, Austria) using optimal gain and a number of 25 flashes.
To examine the saliva composition, 300 ng protein per host race were applied to a polyacrylamide gel. As a marker 1 µL PageRulerTM Plus Prestained Protein Ladder (Thermo Fisher Scientific, Pittsburgh, PA, USA) was utilized. For native gel electrophoresis, the samples were separated on an 8% Tris-glycine gel (InvitrogenTM NovexTM WedgeWellTM; Thermo Fisher Scientific) submerged in a 25 mM Tris, 192 mM glycine running buffer. Native gel electrophoresis was executed with a voltage of 150 V for 1:10 h. For non-native gel electrophoresis, the samples were denatured for 5 min at 90 °C in the presence of SDS loading dye (6x; G-Biosciences, St. Louis, MO, USA) with 2.5% β-mercaptoethanol. SDS-PAGE was executed by separating the proteins on a pre-cast Mini-PROTEAN TGX gel (AnykD, 12 well, 20µL/well; Bio-Rad Laboratories, Feldkirchen, Germany) for 1:10 h at 100 V submerged in running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3).
After gel electrophoresis the gel was fixed by steeping it four times in fixing solution (40% EtOH, 10% acetic acid) for 15 min. Three 20 min washing steps (30% EtOH) were followed by three 10 min rinsing steps (ddH2O). Sensitizer (0.02% Na2S2O3) was applied for one minute and the gel subsequently rinsed three times with ddH2O. The gel was incubated in staining solution (0.2% AgNO3, 0.02% formaldehyde) for 20 min and then rinsed three times. The developer (3% Na2CO3, 0.04% formaldehyde) was added to the gel and immediately removed after the staining was as strong as required. After washing two times with ddH2O, a stop solution (5% acetic acid) was added for 5 min. Gels were stored in 1% acetic acid. Pictures were taken with a Lumix Digital Camera DMC-FZ200 (Panasonic, Wiesbaden, Germany) and figures compiled with Adobe Illustrator CS5.

4.5. Statistics

To investigate the influence of different A. pisum host races on forisome dispersion, the dispersion behavior was categorized as either fully dispersed or not fully dispersed. In case multiple forisomes were observed in one plant, one forisome was selected at random with the help of R. The influence of the A. pisum host race, plant age (both used as fixed explanatory factors) and distance between heat stimulus and observation site (used as fixed continuous explanatory variable) on the ratio of fully dispersed forisomes was analyzed per plant with a Bernoulli generalized linear model (GLM). To determine the p-values of the main explanatory factors, the explanatory factors were removed one after another from the model and the simpler models were compared to the more complex model with an analysis of deviance test [81]. In case of significant differences, factor level reduction was applied to detect differences between treatments [82]. All statistical analyses were done in R version 4.2.1 [83].

Author Contributions

Conceptualization, M.K.P., A.C.U.F. and G.K.; methodology, M.K.P., A.C.U.F. and G.K.; validation, M.K.P. and A.C.U.F.; formal analysis, M.K.P., A.C.U.F. and G.K.; investigation, M.K.P., L.W. and A.C.U.F.; resources, J.G., A.C.U.F. and G.K.; data curation, M.K.P. and G.K.; writing—original draft preparation, M.K.P.; writing—review and editing, M.K.P., A.C.U.F., J.G. and G.K.; visualization, M.K.P., A.C.U.F. and G.K.; supervision, G.K. and A.C.U.F.; project administration, M.K.P., A.C.U.F. and G.K. All authors have read and agreed to the published version of the manuscript.

Funding

A.C.U.F. was supported by the Friedrich-Schiller-University Jena in the frame of ProChanceCareer grant 2.11.3-A1/2020-04. M.K.P., L.W., G.K. and J.G. were funded by the Max Planck Society, Germany. The funder had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability Statement

Data can be obtained upon request from the corresponding authors.

Acknowledgments

We would like to thank Jean-Christophe Simon at INRAE Le Rheu, France for supplying the initial aphid populations and Andreas Weber and Elke Goschala for plant cultivation.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 4. Silver-stained gels of aphid watery saliva. Depicted are (A) a native polyacrylamide gel electrophoresis (PAGE) gel and (B) a denatured sodium dodecyl sulfate (SDS)-PAGE gel. Watery saliva samples were applied analogously to each gel. Lane 1 (M): protein ladder (PageRulerTM Plus Prestained Protein Ladder). The corresponding protein sizes are given in kDa on the left side of each figure panel in lane ‘M’. Lane 2 to 4: For each host race about 300 ng protein was applied to the gel (MR—Medicago host race; PR—Pisum host race, TR—Trifolium host race). The asterisk indicates large proteins detected in the native PAGE. Arrowheads indicate bands that differ between PR and the other two host races.
Figure 4. Silver-stained gels of aphid watery saliva. Depicted are (A) a native polyacrylamide gel electrophoresis (PAGE) gel and (B) a denatured sodium dodecyl sulfate (SDS)-PAGE gel. Watery saliva samples were applied analogously to each gel. Lane 1 (M): protein ladder (PageRulerTM Plus Prestained Protein Ladder). The corresponding protein sizes are given in kDa on the left side of each figure panel in lane ‘M’. Lane 2 to 4: For each host race about 300 ng protein was applied to the gel (MR—Medicago host race; PR—Pisum host race, TR—Trifolium host race). The asterisk indicates large proteins detected in the native PAGE. Arrowheads indicate bands that differ between PR and the other two host races.
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Plants 12 01888 sch001 550
Scheme 1. Forisome dispersion depends on cytosolic Ca2+ concentrations in the sieve elements (SEs). After application of a trigger (heat stimulus or damage), the Ca2+ concentration increases and forisomes (white encircled structures in the micrographs) start to disperse (dispersion threshold; black, dotted line). Due to a change in the structure and light refraction, the forisome is no longer visible with light microscopy and appears smaller. If a certain Ca2+ concentration (red, dashed line) is reached, forisomes (location indicated by asterisk) fully disperse, become invisible with light microscopy, and block the SE. The Ca2+ concentration threshold may vary for plants with different forisome sizes. SE—sieve element; CC—companion cell.
Scheme 1. Forisome dispersion depends on cytosolic Ca2+ concentrations in the sieve elements (SEs). After application of a trigger (heat stimulus or damage), the Ca2+ concentration increases and forisomes (white encircled structures in the micrographs) start to disperse (dispersion threshold; black, dotted line). Due to a change in the structure and light refraction, the forisome is no longer visible with light microscopy and appears smaller. If a certain Ca2+ concentration (red, dashed line) is reached, forisomes (location indicated by asterisk) fully disperse, become invisible with light microscopy, and block the SE. The Ca2+ concentration threshold may vary for plants with different forisome sizes. SE—sieve element; CC—companion cell.
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Figure 5. Representative infested experimental plants of the different age groups used. Arrowheads indicate the leaflets used for experiments. Presented are examples of (A) Vicia faba plants with the red-colored Trifolium host race (ID210), (B) Trifolium pratense with the green-colored Medicago host race (ID218), and (C) Pisum sativum with the green-colored Pisum host race (ID212).
Figure 5. Representative infested experimental plants of the different age groups used. Arrowheads indicate the leaflets used for experiments. Presented are examples of (A) Vicia faba plants with the red-colored Trifolium host race (ID210), (B) Trifolium pratense with the green-colored Medicago host race (ID218), and (C) Pisum sativum with the green-colored Pisum host race (ID212).
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Table
Table 2. Putative identity of proteins in pea aphid saliva based on the apparent molecular weight after SDS-PAGE. We matched our findings with the results of Carolan et al. 2009 [39] and 2011 [49], which investigated saliva from MR aphids. The second column indicates which pea aphid host races contained bands of the respective molecular weights as demonstrated by electrophoresis in this study.
Table 2. Putative identity of proteins in pea aphid saliva based on the apparent molecular weight after SDS-PAGE. We matched our findings with the results of Carolan et al. 2009 [39] and 2011 [49], which investigated saliva from MR aphids. The second column indicates which pea aphid host races contained bands of the respective molecular weights as demonstrated by electrophoresis in this study.
Apparent Molecular WeightHost Races in Which Band DetectedEST Cluster or Scaffold 1Putative Protein Identification 1A. pisum Reference Sequence 1
190 kDaMR, PR, TRUnknownNANA
150 kDaMR, PR, TRAPG02147Putative sheath protein 2XM001943863 (ACYPI009881)
APG01291M1 zinc metalloproteaseXM001950011 (ACYPI009427)
130 kDaNAAPG02147Putative sheath protein 2XM001943863 (ACYPI009881)
APG01291M1 zinc metalloproteaseXM001950011 (ACYPI009427)
115 kDaMR, PR, TRAPG09617Angiotensin converting enzyme (ACE)XM001951605 (ACYPI000733)
APG09831XM001944530 (ACYPI008911)
100 kDaMR & TRAPG09617XM001951605 (ACYPI000733)
APG09831XM001944530 (ACYPI008911)
80 kDaMR, PR, TRNANANA
66 kDaMR, PR, TRAPG028784No informationNo information
55 kDaMRNo EST matchNANA
41 kDaMRAPG10010SMP-30 (regucalcin)XM001951513 (ACYPI003308)
NA—not applicable; MR—Medicago host race; PR—Pisum host race; TR—Trifolium host race. 1—as published by Carolan et al. 2009 [39]; 2—as published by Carolan et al. 2011 [49].
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Paulmann, M.K.; Wegner, L.; Gershenzon, J.; Furch, A.C.U.; Kunert, G. Pea Aphid (Acyrthosiphon pisum) Host Races Reduce Heat-Induced Forisome Dispersion in Vicia faba and Trifolium pratense. Plants 2023, 12, 1888. https://doi.org/10.3390/plants12091888

AMA Style

Paulmann MK, Wegner L, Gershenzon J, Furch ACU, Kunert G. Pea Aphid (Acyrthosiphon pisum) Host Races Reduce Heat-Induced Forisome Dispersion in Vicia faba and Trifolium pratense. Plants. 2023; 12(9):1888. https://doi.org/10.3390/plants12091888

Chicago/Turabian Style

Paulmann, Maria K., Linus Wegner, Jonathan Gershenzon, Alexandra C. U. Furch, and Grit Kunert. 2023. "Pea Aphid (Acyrthosiphon pisum) Host Races Reduce Heat-Induced Forisome Dispersion in Vicia faba and Trifolium pratense" Plants 12, no. 9: 1888. https://doi.org/10.3390/plants12091888

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