Modified expression of alternative oxidase in transgenic tomato and petunia affects the level of tomato spotted wilt virus resistance

Abstract

Background: Tomato spotted wilt virus (TSWV) has a very wide host range, and is transmitted in a persistent manner by several species of thrips. These characteristics make this virus difficult to control. We show here that the over-expression of the mitochondrial alternative oxidase (AOX) in tomato and petunia is related to TSWV resistance.

Results: The open reading frame and full-length sequence of the tomato AOX gene LeAox1au were cloned and introduced into tomato 'Healani' and petunia 'Sheer Madness' using Agrobacterium-mediated transformation. Highly expressed AOX transgenic tomato and petunia plants were selfed and transgenic R1 seedlings from 10 tomato lines and 12 petunia lines were used for bioassay. For each assayed line, 22 to 32 tomato R1 progeny in three replications and 39 to 128 petunia progeny in 13 replications were challenged with TSWV. Enzyme-Linked Immunosorbent Assays showed that the TSWV levels in transgenic tomato line FKT4-1 was significantly lower than that of wild-type controls after challenge with TSWV. In addition, transgenic petunia line FKP10 showed significantly less lesion number and smaller lesion size than non-transgenic controls after inoculation by TSWV.

Conclusion: In all assayed transgenic tomato lines, a higher percentage of transgenic progeny had lower TSWV levels than non-transgenic plants after challenge with TSWV, and the significantly increased resistant levels of tomato and petunia lines identified in this study indicate that altered expression levels of AOX in tomato and petunia can affect the levels of TSWV resistance.

Figure 1

Constructs developed for plant transformations. PCR amplicons of the ORF and full-length LeAox1au gene were cloned into pBI525 at the XbaI and BamHI sites (dashed line). The resulting fragment was excised from pBI525 at the Hind III and EcoR I sites and ligated into pBI121 to form the transformation constructs.

Figure 2

Southern blot analyses of selected transgenic tomato plants. Genomic DNA was digested with EcoR I, separated in 1% agarose gel, transferred to a nylon membrane, and hybridized to a 620 bp DIG-labeled probe of the 35S promoter sequence. Numbers on the left are approximate sizes in kilobase pairs. Lanes 1 to 10: 1, FDT3-3-1; 2, FDT3-1; 3, CDT1-1; 4, CDT13; 5, CDT17-1; 6, CDT17-3; 7, CKT1; 8, CKT6; 9, 525DT5; 10, wild type.

Figure 3

Northern blot analyses of selected transgenic tomato plants. Panel A, total RNA prepared from tomato plants was probed with the ORF fragment of LeAox1au. Panel B, tomato 18S rRNA. Lanes 1 to 17: 1, FDT3-3-1; 2, FDT3-3-2; 3, FDT3-1; 4, FDT3-3-3; 5, FKT6; 6, FKT7; 7, CDT1-1, 8, CDT6; 9, CDT10; 10, CDT13; 11, CDT16-1; 12, CDT17-1; 13, CDT17-3; 14, CKT1; 15, CKT6; 16, 525DT5; 17, wild type. The values below panel B are the ratios of the intensity of hybridization signal of the sample in panel A verses that in panel B.

Figure 4

Western blot analyses of AOX expression of some tomato transgenic and wild type lines. A, mitochondrial proteins isolated from transgenic lines CKT1, FDT3-3-1, FDT3-3-3 and non-transgenic control plants were separated in 12% SDS-PAGE gels, transferred to PVDF membranes, probed with mouse monoclonal antibody AOA and detected with NBT/BCIP. B, mitochondrial protein stained with Coomassie. U, upper leaf; M, middle leaf; L, lower leaf.

Figure 5

Mean ELISA readings of progeny from 2 transgenic tomato lines and wild-type control plants. Two lines of transgenic tomato (FKT9 and FKT4-1) and wild-type control plants were inoculated twice with TSWV and virus titer assayed by ELISA at different time points over a 47 day period.