Department of Plant Biology, Genetics Centre, SLU, PO Box 7080, S-750 07 Uppsala, Sweden1
Author for correspondence: Jari Valkonen. Fax +46 18 67 3392. e-mail jari.valkonen{at}vbiol.slu.se
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Since virus resistance obtained in transgenic plants is usually virus-specific and often based on gene silencing, infection of the plants with other viruses could reduce levels of resistance due to suppression of gene silencing. However, this possibility remains to be tested. Therefore, the aim of this study was to generate CP gene-mediated resistance to Potato virus A (PVA, genus Potyvirus) in transgenic plants, and to test whether the resistance to PVA was affected by infection of the transgenic plants with PVY, another potyvirus known to suppress gene silencing (Brigneti et al., 1998 ; Mäki-Valkama et al., 2000
).
Nicotiana benthamiana was transformed with the CP gene of PVA (isolate B11) driven by the 35S promoter of Cauliflower mosaic virus fused with the 5'-untranslated region of the PVA genome to enhance transgene expression. N. benthamiana transformed with the glucuronidase (GUS) marker gene under control of the 35S promoter was used as a control in virus experiments. The constructs and plant materials as well as the experimental conditions and protocols used in this study have been described elsewhere in detail (Savenkov & Valkonen, 2001 ). Thirteen CP-transgenic lines were obtained, and all were resistant to PVA-B11, as indicated by absence of detectable infection following mechanical inoculation (data not shown). The transgenic lines were self-pollinated and the T1 progeny of four CP-transgenic lines were tested for PVA resistance. Some of these T1 progenies contained a few non-transgenic segregants which were susceptible to PVA, whereas all transgenic T1 progeny were PVA-resistant, similar to the primary transformants (data not shown). The T1 progeny of one CP-transgenic line (line ab10) and one GUS-transgenic line that do not segregate for the transgene were selected for this study. Plants were grown from seed under conditions described elsewhere (Savenkov & Valkonen, 2001
).
The upper leaves of uninoculated, 4-week-old plants of line ab10 were tested for expression of PVA CP and the CP-transgene mRNA. CP was tested by DAS-ELISA (Savenkov & Valkonen, 2001 ) and Western analysis (Sambrook et al., 1989
) using monoclonal antibodies specific to PVA CP and including known amounts of purified virions of PVA for comparison. Development of reaction with substrate was terminated at the time-point when the lowest amount of virions (0·16 ng per well) had reached an ELISA absorbance value (A405) of 0·20 (for buffer wells and GUS-transgenic plants, A405=0·00). No elevation of A405 values was observed for samples from the transgenic plants (values similar to buffer wells and samples from the GUS-transgenic plants) and no CP was detectable by Western analysis. Total RNA was extracted from the leaves and subjected to Northern analysis using the 32P-labelled PVA CP cDNA as a probe, but no signal corresponding to the CP-transgene mRNA was observed. However, RTPCR carried out on the total RNA (treated with DNase) using PVA CP gene-specific primers produced a product of the expected size from plants of line ab10. No amplification was obtained from the DNase-treated RNA without RT prior to PCR, or from total RNA of the control (GUS-transgenic) plants. These data indicate that the steady-state expression levels of the PVA CP gene were very low, in contrast to expression of GUS mRNA, which was readily detectable in the GUS-transgenic plants (using the histochemical
-glucuronidase assay).
Four-week-old plants of line ab10 and the GUS-transgenic line were mechanically inoculated with PVA-B11 (inoculum concentration ca. 1 µg virions/ml sterile distilled water) the day after samples had been taken for analysis of transgene expression. Two leaves per plant of a total of 10 plants per line were inoculated in three experiments. The inoculated and upper non-inoculated leaves were tested for PVA by DAS-ELISA at 14 days post-inoculation (p.i.). In plants of line ab10, neither the inoculated leaves nor the upper non-inoculated leaves were infected with PVA (A405 values no different from buffer wells) and plants were symptomless. In contrast, the plants of the GUS-transgenic line developed symptoms of severe mosaic and leaf malformation at 810 days p.i., and all plants were systemically infected with PVA (>8000 ng PVA CP antigen/g leaf, as determined by ELISA). Sap was extracted from the PVA-inoculated leaves of line ab10, diluted fivefold with distilled water and used for inoculation of healthy seedlings of the GUS-transgenic line, but no inoculated plant was infected at 14 days p.i. These results indicated that line ab10 expressed high levels of CP gene-mediated resistance to PVA, and that no detectable infection occurred in the inoculated plants.
In the next experiment, 25 plants of line ab10 were grown for 2 weeks, and then five plants were sap-inoculated with PVY (isolate UK, ordinary strain group of PVY). Symptoms of severe mosaic and leaf malformation developed in the PVY-inoculated plants at 810 days p.i. The symptomatic leaves were tested for PVY by DAS-ELISA using polyclonal antibodies to PVY (Adgen) and including known amounts of purified virions of PVY for comparison. All symptomatic leaves contained high amounts of the PVY CP antigen (>2000 ng/g). These data indicated that the PVA CP gene provided no resistance to PVY in the transgenic line ab10. Taken together, the very low steady-state levels of transgene mRNA, the high levels of resistance to the homologous virus, and the lack of resistance to a heterologous virus suggested that the resistance to PVA in line ab10 was based on homology-dependent gene silencing (Marathe et al., 2000 ).
Total RNA was extracted from the upper leaves of the healthy plants and PVY-infected plants (14 days p.i.) of line ab10 and subjected to quantitative Northern blot analysis for detection of the PVA CP-transgene mRNA. As before, its expression in healthy plants of line ab10 was extremely low or below the detection limit even using a long exposure of the film, whereas it was readily detectable in the PVY-infected plants (Fig. 1). Hence, infection with PVY led to reversal of the PVA CP gene silencing in the transgenic plants, as revealed by the resumption of transgene mRNA accumulation.
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This study has shown that high levels of resistance to PVA were expressed in N. benthamiana transformed with the CP-encoding sequence of PVA. The observed resistance was associated with very low steady-state levels of the transgene mRNA. These findings are consistent with some of the first reports on the CP gene-mediated resistance to potyviruses (Lawson et al., 1990 ; Lindbo & Dougherty, 1992
; Lindbo et al., 1993
). While this study describes the first example of CP gene-mediated resistance to PVA, many previous studies have reported similar resistance to other potyviruses (reviewed in Lomonossoff, 1995
; Mäki-Valkama & Valkonen, 1999
). Resistance to PVA was homology-dependent and did not protect the plants against infection with PVY (comparison of the CP-encoding region of PVA-B11 to those of 13 PVY isolates retrieved from databases reveals only 5356% and 5860% identity at the nucleotide and amino acid sequence level, respectively). Consequently, the steady-state levels of the CP gene mRNA were greatly increased in the plants infected with PVY, which is consistent with suppression of gene silencing (Marathe et al., 2000
). Therefore, this situation was reminiscent of a previous study where silencing of the green fluorescent marker gene (GFP) in transgenic plants could be suppressed by ectopic expression of the HC-Pro of PVY, resulting in readily detectable expression of GFP (Brigneti et al., 1998
).
The important and novel finding of this study is that CP gene-mediated virus resistance in transgenic plants may be suppressed by infection of the plants with heterologous viruses that encode suppressors of gene silencing, such as the HC-Pro of PVY. In contrast to healthy plants of the CP gene-transgenic lines, in which no detectable infection with PVA was observed following challenge with a high PVA inoculum dose, all the PVY-infected plants of the same transgenic line were readily infectible with PVA and accumulated moderate titres of PVA CP and RNA in the systemically infected leaves. Since PVY and PVA share only a low level of sequence similarity (see above), cross-protection between these viruses does not occur, and does not prevent superinfection of the PVY-infected plants with PVA. Hence, the practical impact of the negative effects of gene silencing suppression on transgenic virus resistance in the field will depend on the availability of virus sources and vectors determining the chance of co-infections of plants.
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References |
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Received 4 April 2001;
accepted 30 May 2001.