This study tried to address two issues: 1) what is the nature of BNYVV association with its vector; and 2) how is virus transferred between the vector and root cells. Using immunofluorescence and immunogold labeling techniques, all BNYVV proteins were detected in P. betae resting spores and zoospores. Samples were also treated with buffer or heterologous antiserum (Figure 2) and these produced negative results, indicating that the immunolabelling was specific for BNYVV proteins. Comparing the immunogold label in BNYVV and healthy P. betae zoosporangia, provides further evidence that each BNYVV protein occurs inside virus containing P. betae zoosporangia.
There are two possible explanations for detection of all BNYVV proteins inside P. betae sporosori and zoosporangia. The first model, proposed by Campbell, suggests that the Polymyxa and plant cytoplasms have opportunities to mix and that virus may be freely exchanged on these occasions. Campbell proposed that this may occur before membranes are laid down to form the sporangial plasmodium . The data presented in this paper support this model. Furthermore, we found occasional breaks in the zoosporangial wall which could create additional opportunities for the P. betae and plant cell cytoplasms to mix. If BNYVV is replicating in the same plant cell that is infected with P. betae, then it is reasonable to consider that all BNYVV proteins move freely between P. betae when there are breaks in the zoosporangial wall as well as during developmental of the sporangial plasmodium.
An alternative explanation is that P. betae is a host as well as a vector for BNYVV. The presence of viral replicase inside P betae resting spores and zoospores may be evidence that BNYVV replicates inside its vector. According to this model, accumulation of viral replicase, coat protein, RTD, P25, and P31 proteins, which are expressed from the genomic RNAs, suggests that viral RNAs may be translated as P. betae progresses through its life cycle. The P42, P13, P15, and P14 proteins are produced from subgenomic RNAs derived from BNYVV RNA2. Subgenomic RNA expression is dependent on production of minus strand RNAs involved in virus replication.
Considering the P. betae life cycle (Figure 1B), it is reasonable to consider that the spread of BNYVV within the developing zoosporangial thallus into the innumerable secondary zoospores, requires the virus to multiply within its vector. Similarly, following penetration of secondary zoospores into plant cells, the developing plasmodium may take up virus from the plant cell cytoplasm. However, virus multiplication is likely for BNYVV to spread within the developing sporosorus and most of the resting spores. Immunolabelling of sporosori and zoosporangia, which represent the sporogenic and sporangial stages of the P. betae life cycle, showed that each BNYVV protein associates with both life cycle stages. This could be evidence of different cytoplasms mixing or evidence that BNYVV multiplies inside its vector.
Tables 1 and 2 compare the subcellular distribution of the BNYVV proteins to determine if they localize to specific regions of the spores. In resting spores, the concentration of viral replicase was greatest in the cell wall, but was significant in most structures. Each of the BNYVV proteins had distinct subcellular accumulation patterns in resting spores, making it difficult to identify specific centers for viral activities. In zoospores, most of the BNYVV proteins are greatest in the cytoplasm, vesicles, and areas between spores. If BNYVV replicates inside P. betae zoospores, then these data could be explained by two different models (Figure 1C). Possibly, the BNYVV proteins are translated on free ribosomes in the cytoplasm, and are captured into secretory vesicles for virus replication and packaging. Virions, viral replication complexes, or viral movement complexes are then carried to the exterior of the cell. Alternatively the flow of events is in the other direction: virus may be captured from the cell exterior by pinocytotic vesicles. Virions may disassemble in the vesicles and viral RNA is released in the cytoplasm for translation and replication (Figure 3G and 4A).
The subcellular accumulation patterns for BNYVV P25 and P31 were intriguing because these proteins are suggested to play significant roles in virus transmission and accumulation in roots [23, 27]. Here we show that both proteins accumulate to significant levels in P. betae. In this study P25 and P31 are the only proteins that localize to the zoospore nucleus. Prior studies in plant cells also show P25 traffics to the nucleus . If P25 and P31 are actively transported into the nucleus, this would suggest that they are actively interacting with cellular components and that they may be functional within the P. betae zoospore. It has been suggested that nuclear accumulation of P25 may play a role in symptom expression in plants . It would be intriguing to learn if P25 has similar abilities to cause disease symptoms in P. betae.
Further investigations are needed to determine if P. betae is a host for BNYVV. This requires developing research tools to study the time course of viral RNA accumulation in P. betae sporosori and zoosporangia. While there are examples of plant RNA viruses which multiply inside their insect vectors, and plant viruses that can replicate in yeast, there are no examples yet of plant viruses replicating inside plasmodiophorid vectors. BNYVV requires the RTD to mediate vector transmission to plants. This study revealed that there is an opportunity for more than one BNYVV protein to participate in vector acquisition and transmission. As we develop more tools for studying the association of BNYVV viruses with its vector, we will learn if transmission is actively enabled by specific viral proteins, or is the result of passive mixing of two cytoplasms as suggested by Campbell .