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

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.

Keywords: Pisum sativum; Trifolium pratense; Vicia faba; aphid saliva; calcium; legume; pea aphid host race; phloem located defense; phloem protein; sieve element occlusion.

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 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 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 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 (PageRuler^TM 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.

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.

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).