Tuula Pehu

Potato plants transformed with a potato virus Y P1 gene sequence are resistant to PVYO

The cloned P1 sequence of PVYO was transferred in sense orientation into the potato cultivar Pito usingAgrobacteriummediated transformation. Sixteen of the putatively transformed plants (NPTII positive) were assayed for PVYO resistance. No PVYO was detected in four plants, representing two lines, 21 days after two sap-inoculations and 35 days after graft-inoculation, and the plants remained symptomless, whereas other tested plants showed mosaic symptoms and had high PVY titers similar to those of the control plants. No line was resistant to PVYN and potato viruses A and X. Southern analysis confirmed the presence of the transgene(s) in the two PVYO-resistant and one susceptible line examined, but no signal was detected in nontransformed Pito. These results suggest a high level of protection against PVYO in potato transformed with P1 sequence of PVYO.CompendioLa cadena clonada del PI de PVYO fue transferida, en orientatión positiva, al cultivar Pito de papa, utilizando la transformatión mediante Agrobacterium. Dieciséis de las plantas transformadas putativamente (NPTII positivo) fueron evaluadas para resistencia a PVYO. No se detectó PVYO en cuatro plantas, representando dos líneas, 21 días después de dos inoculaciones con savia y 35 días después de inoculatión por injerto, y las plantas permanecieron sin síntomas, mientras que otras de las plantas probados mostraron síntomas de mosaico y tuvieron titulaciones altas de PVYsimilares a aquellas de las plantas testigo. Ninguna línea fue resistente al PVYN o a los virus A y X de la papa. El análisis del RNA en placas de nitrocelulosa confirmó la presencia del (o de los) transgene (s) en las dos líneas resistentes y en una susceptible al PVYO examinadas, pero no se detectó señal alguna en las plantas de Pito no transformadas. Estos resultados sugieren un alto nivel de protección contra el PVYO en la papa transformada con el encadenamiento del PI de PVYO.

Localization of the P1 protein of potato Y potyvirus in association with cytoplasmic inclusion bodies and in the cytoplasm of infected cells.

The N-terminal P1 proteinase of potato virus Y (ordinary strain group isolate PVY-O) was expressed in E. coli. Antiserum was raised against the expressed protein and used to detect the viral proteins in infected tobacco leaf tissue by Western blotting and by electron microscopy with immunogold labelling. In the immunogold localization studies P1 protein was detected in association with the cytoplasmic inclusion bodies characteristic of PVY infections and in the cytoplasm of the infected plant cells. No significant P1 antibody binding with other plant cell organelles, or with the cell wall and plasmodesmata, was detected by immunogold labelling.

High level of resistance to potato virus Y by expressing P1 sequence in antisense orientation in transgenic potato

Potato cultivar Pito was transformed with the P1 sequence of potato virus Y (PVYO) in antisense orientation. Five transgenic lines (ASR) showed a high level of resistance to PVYO on mechanical and side-graft inoculation, expressed as no symptoms, and no detectable amounts of PVY in the inoculated and the upper uninoculated leaves, as determined by ELISA 21 and 35 days after inoculation. The possibility of accumulation of low levels of PVYO not detected by ELISA was tested by grafting a scion of untransformed Pito (WT) on each of the PVYO-inoculated ASR plants. With this method, no PVY was detected in the WT plants grafted on the PVYO-inoculated ASR plants. Two of the six plants of the ASR line AI 0623-2 showed PVY accumulation when grafted on the PVYO-inoculated WT plants. Resistance was specific to PVYO, as the ASR lines were fully susceptible to PVYN, PVA and PVX. The ASR lines and PVY-susceptible lines contained 1–5, and 1–3 P1 gene inserts, respectively, as determined by Southern analysis. All lines expressed similarly low levels of the antisense P1 transcript. These results show that effective, virus strain-specific resistance to PVYOcan be achieved by expressing the P1 sequence in antisense orientation in potato. The low transgene transcript levels suggest that resistance in the ASR plants may be based on gene silencing.