Characterization of horizontal transmission of blueberry latent spherical virus by pollen.

Nicotiana benthamiana plants became infected with blueberry latent spherical virus (BLSV) after pollination with pollen grains produced by BLSV-infected N. benthamiana plants. Interestingly, pollen grains produced by BLSV-infected Vaccinium corymbosum (blueberry), Nicotiana alata, and Petunia × hybrida (petunia) plants also transmitted the virus to healthy N. benthamiana plants after pollination. As seen using aniline blue staining and fluorescence microscopy, pollen grains from BLSV-infected blueberry, N. alata, and petunia plants germinated on stigmas of N. benthamiana, and the pollen tubes penetrated the stigmas in a manner similar to that of N. benthamiana pollen grains on N. benthamiana stigmas. Whole-mount in situ hybridization and chromogenic in situ hybridization analysis showed that infected blueberry and N. benthamiana pollen grains germinated on N. benthamiana stigmas, and virus-containing pollen tubes penetrated the stigmas. Tissue blot hybridization analysis revealed that the initial infection sites were the N. benthamiana stigmas pollinated with infected pollen grains from blueberry and N. benthamiana. In addition, the virus spread from the initial infection sites to the phloem in the stigma and style. Taken together, we suggest that penetrating pollen tubes that harbored the virus results in infection foci in the stigma, and the virus then moves to the vascular tissues in the stigma and style and eventually establishes systemic infection.

Horizontal pollen transmission of Gentian ovary ring-spot virus is initiated during penetration of the stigma and style by infected pollen tubes.

Gentian ovary ring-spot virus (GORV) infected gentian plants by pollination with GORV-infected gentian pollen grains, but the virus was not horizontally transmitted to gentian plants by transfer of pollen from GORV-infected Nicotiana benthamiana plants. However, N. benthamiana plants were infected with the virus by pollination with infected gentian pollen as well as by pollination with infected N. benthamiana pollen. When infected gentian pollen grains were placed on N. benthamiana stigmas, germinating pollen tubes penetrated into the stigmas and the styles (stigma-style). Virus infection occurred during penetration of the stigma-style, and the virus subsequently spread systemically to the mother plant. On the other hand, most infected N. benthamiana pollen grains failed to germinate on gentian stigmas, and virus infections were not detected in the stigma-style.

Pollen tubes introduce Raspberry bushy dwarf virus into embryo sacs during fertilization processes.

We developed a fertilization method in which pollen tubes entered into embryo sacs without any need to contact surrounding female sporophytic cells by using Torenia fournieri (Torenia) plants under the condition of hindering movement of the virus from a stigma, which is the first infection site leading to systemic infection. When RBDV-infected Torenia pollen grains were used for the developed fertilization method, the virus was transmitted to the seeds by pollen tubes germinating from them. On the other hand, no seeds were infected with the virus when Torenia plants were pollinated with healthy Torenia pollen grains in combination with RBDV-infected raspberry pollen grains, which caused the virus infection in the stigma by penetration of their pollen tubes arrested in its style. Our results indicate that vertical transmission of RBDV by pollen occurs in the transport of the virus into embryo sacs by pollen tubes reaching the embryo sacs.

Penetration of pollen tubes with accumulated Raspberry bushy dwarf virus into stigmas is involved in initial infection of maternal tissue and horizontal transmission.

Torenia fournieri (Torenia) plants were infected with Raspberry bushy dwarf virus (RBDV) by pollination with RBDV-infected raspberry pollen grains. The infected raspberry pollen grains germinated on Torenia stigmas, and then the pollen tubes penetrated into the stigma, even though the pollen tubes were arrested in the styles. In whole-mount in situ hybridization of germinating infected raspberry pollen grains, RBDV accumulated in the tips of the pollen tubes. Tissue blot hybridization of Torenia plants pollinated with infected raspberry pollen grains revealed that the first virus infection site leading to systemic infection is the stigma. When infected raspberry pollen grains that had lost germination capacity were pollinated on Torenia stigmas, RBDV could not infect the stigmas, and no horizontal transmission occurred. These results indicate that penetration of pollen tubes with accumulated RBDV into stigmas is essential in causing the first viral infection in the stigma to lead to systemic infection.