A TOM1 homologue is required for multiplication of Tobacco mosaic virus in Nicotiana benthamiana

Abstract

The AtTOM1 gene of Arabidopsis thaliana had been shown to be essential for the efficient multiplication of Tobacco mosaic virus (TMV) in A. thaliana. In this study, we cloned an AtTOM1-like gene from Nicotiana benthamiana named as NbTOM1. Sequence alignment showed that NbTOM1 is closely related to AtTOM1 homologues of N. tabacum and Lycopersicon esculentum with 97.2% and 92.6% nucleotide sequence identities, respectively. Silencing of NbTOM1 by a modified viral satellite DNA-based vector resulted in complete inhibition of the multiplication of TMV in N. benthamiana. The result suggests that inhibition of NbTOM1 via RNA silencing is a potentially useful method for generating TMV-resistant plants.

Fig. 1

Effects of the knockdown of NbTOM1 expression on TMV accumulation in N. benthamiana. Plants were pre-inoculated with TYLCCNV and DNAmβ: NbTOM1 (a), TYLCCNV and DNAmβ empty vector (b) and water (c), respectively, and then challenge inoculated with the TMV:GFP clone 15 d after pre-inoculation. The NbTOM1 silenced plant appear red due to non-proliferation of TMV:GFP (a). The NbTOM1 non-silenced control plant appear green due to GFP expression resulting from TMV:GFP accumulation (b, c). Photographs were taken under UV light 3 weeks after challenge inoculation

Fig. 1

Effects of the knockdown of NbTOM1 expression on TMV accumulation in N. benthamiana. Plants were pre-inoculated with TYLCCNV and DNAmβ: NbTOM1 (a), TYLCCNV and DNAmβ empty vector (b) and water (c), respectively, and then challenge inoculated with the TMV:GFP clone 15 d after pre-inoculation. The NbTOM1 silenced plant appear red due to non-proliferation of TMV:GFP (a). The NbTOM1 non-silenced control plant appear green due to GFP expression resulting from TMV:GFP accumulation (b, c). Photographs were taken under UV light 3 weeks after challenge inoculation

Fig. 1

Effects of the knockdown of NbTOM1 expression on TMV accumulation in N. benthamiana. Plants were pre-inoculated with TYLCCNV and DNAmβ: NbTOM1 (a), TYLCCNV and DNAmβ empty vector (b) and water (c), respectively, and then challenge inoculated with the TMV:GFP clone 15 d after pre-inoculation. The NbTOM1 silenced plant appear red due to non-proliferation of TMV:GFP (a). The NbTOM1 non-silenced control plant appear green due to GFP expression resulting from TMV:GFP accumulation (b, c). Photographs were taken under UV light 3 weeks after challenge inoculation

Fig. 2

Semi-quantitative RT-PCR analysis showing the effect of VIGS with DNAmβ:NbTOM1 in N. benthamiana. Lanes 1~6 correspond to products from PCR cycle Nos. 15, 18, 21, 24, 27 and 30. Lane M represents size markers. (a) PCR products for EF-1-α derived from N. benthamiana co-agroinfected with TYLCCNV and DNAmβ:NbTOM1 (left) or with TYLCCNV and DNAmβ empty vector (right). Gene EF-1-α served as an internal control for RNA quantity in RT-PCR; (b) PCR products of NbTOM1 from N. benthamiana co-agroinoculated with TYLCCNV and DNAmβ: NbTOM1 (left) or with TYLCCNV and DNAmβ empty vector (right)

Fig. 3

Northern blot analysis of TMV accumulation in NbTOM1 silenced and non-silenced N. benthamiana plants at 18 d post-inoculation with TMV:GFP Lanes 1 and 2 show viral RNA from NbTOM1-non-silenced plants; Lanes 3 and 4 show viral RNA from individual NbTOM1-silenced plants. Lower panels show RNAs stained with ethidium bromide