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.