Intracellular distribution of viral gene products regulates a complex mechanism of cauliflower mosaic virus acquisition by its aphid vector

Abstract

Interactions between Cauliflower mosaic virus (CaMV) and its aphid vector are regulated by the viral protein P2, which binds to the aphid stylets, and protein P3, which bridges P2 and virions. By using baculovirus expression of P2 and P3, electron microscopy, surface plasmon resonance, affinity chromatography, and transmission assays, we demonstrate that P3 must be previously bound to virions in order that attachment to P2 will allow aphid transmission of CaMV. We also show that a P2:P3 complex exists in the absence of virions but is nonfunctional in transmission. Hence, unlike P2, P3 and virions cannot be sequentially acquired by the vector. Immunogold labeling revealed the predominance of spatially separated P2:P3 and P3:virion complexes in infected plant cells. This specific distribution indicates that the transmissible complex, P2:P3:virion, does not form primarily in infected plants but in aphids. A model, describing the regulating role of P3 in the formation of the transmissible CaMV complex in planta and during acquisition by aphids, is presented, and its consequences are discussed.

Figure 1

Differential localization of P2 and P3 in infected turnip leaf mesophyll cells. (A) Overview of a mesophyll cell showing electron-lucent (el) and electron-dense (ed) viral inclusion bodies. Immunogold double-labeling of an edIB (B) and an elIB (C) shows that P2 (30 nm gold) is detected exclusively in elIBs, whereas P3 (10 nm gold) is found in both types of inclusions. The arrows point to virus particles. Bar = 0.6 μm (A) and 0.3 μm (B and C).

Figure 2

P2 and P3 form aggregates in baculovirus-infected Sf9 cells. Coinfection of Sf9 cells with recombinant baculoviruses encoding P2 and P3 results in formation of inclusions (A) that are (B) labeled by P2 (30 nm gold) and P3 (10 nm gold) antisera whereas P3 in singly infected Sf9 cells does not form inclusions (C) but (D) gives rise to a uniform P3 label (10 nm gold). Bar = 1 μm (A and C) and 0.5 μm (B and D); cyt, cytoplasm; n, nucleus.

Figure 3

Affinities between P2, P3, and virions. Solutions containing test proteins were passed over virions, HP2, or HP2-C_ter immobilized on Biacore sensor chips. The change in mass of the sensor chip because of binding/dissociation of protein was recorded in real-time as change in surface plasmon resonance in response units (RU). (A) Increasing concentrations of P3 (5, 10, 20, 40, and 80 μg/ml; tracks 1–5) were injected on immobilized virions. (B) Increasing concentrations of P3 (4, 8, 16, and 32 μg/ml) together with an excess amount of virions (150 μg/ml; tracks 1–4) were injected on immobilized HP2. In track 5, 32 μg/ml P3 was injected on HP2 in the absence of virus; (C) 32 μg/ml P3 was injected on immobilized HP2-C_ter. All injections were started at t = 0 s and stopped at t = 180 s. The control sensorgrams (no protein immobilized) were subtracted from the measuring sensorgrams illustrated.

Figure 4

Reconstitution of the transmissible complex. HP2-charged resin was incubated with virus particles and P3 (lanes 1), virus particles alone (lanes 2), or with P3 alone (lanes 3). The figure shows immunoblots of the flow-through fractions (first three lanes), the last wash fractions (next three lanes), and of the resin (last three lanes) that were analyzed with antisera directed against virus particles (Top), P3 (Middle), and P2 (Bottom). Positions of marker proteins, in kDa, are indicated at the right.

Figure 5

Model of sequential acquisition of CaMV by aphids from infected cells. (A) In infected plant cells, the viral components involved in transmission are spatially separated in elIBs (Left structure) and edIBs (Right structure). Whereas most virus particles (open circles) complexed with P3 (blue bars) are stored in edIBs, P2 (red triangles) in association with P3 and a few virus particles are located in elIBs. When an aphid stylet (at the left) pierces the plasma membrane, saliva is injected into the plant cell. (B) After salivation, the aphid ingests some of the plant cell's contents through its stylet and together with viral inclusion bodies. If an elIB is taken up, it disintegrates and sets free its components P2, P3, and some P2:P3:virion complexes. (C) Although the liberated P3 is lost, the released P2, together with a few P2:P3:virion complexes from the elIB, attaches to the aphid stylet cuticle. The aphid is now P2-loaded, thus, transmission-competent, and ready to acquire more P3:virions (filled circles) from either the same cell or (D) in subsequent punctures, from another cell(s). This model has also been published in a compendium (36).