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We report the application of the hairpin-mediated RNA silencing technology for obtaining resistance to Plum pox virus (PPV) infection in Nicotiana benthamiana plants. Four sequences, covering the P1 and silencing suppressor HC-Pro genes of an Italian PPV M isolate, were introduced into N. benthamiana plants as two inverted repeats separated by an intron sequence under the transcriptional control of the Cauliflower Mosaic Virus 35S promoter. In a leaf disk infection assay, 38 out of 40 T0 transgenic plants were resistant to PPV infection. Eight lines, 2 for each construct, randomly selected among the 38 resistant plants were further analysed. Two hundred forty eight out of 253 T1 transgenic plants were resistant to local and systemic PPV infection. All transgenic single locus lines were completely resistant. These data indicate that the RNA silencing of PPV P1/HCPro sequences results in an efficient and predictable PPV resistance, which may be utilized in obtaining stone fruit plants resistant to the devastating Sharka disease.

A truncated version of the C1 gene of tomato yellow leaf curl geminivirus (TYLCV), encoding the first 210 amino acids of the multifunctional Rep protein, was introduced by Agrobacterium transformation into Lycopersicon esculentum cv. Moneymaker plants under the transcriptional control of an enhanced cauliflower mosaic virus 35S promoter. One R0 plant (line 47) carrying the C1 gene in two loci (A and B) and accumulating the truncated Rep protein (T-Rep), was crossed with either a wild-type plant, or a C1 antisense plant (line 10). The wild type (wt) × 47 progeny were phenotypically homogeneous, contained either A or B locus, expressed high levels of T-Rep protein, had a “curled” phenotype, and were resistant to TYLCV when challenged either by agroinfection or by the vector Bemisia tabaci. In the 10 × 47 progeny, plants carrying only the sense gene behaved like the wt × 47 progeny, while those containing both sense and antisense transgenes did not accumulate the T-Rep protein, showed a normal phenotype, and were not resistant, showing that accumulation of T-Rep protein is required to confer TYLCV resistance. Plants accumulating T-Rep were susceptible to a distinct geminivirus, tomato leaf curl virus (ToLCV-Au).

No abstract

The C1 gene of tomato yellow leaf curl geminivirus (TYLCV) encodes a multifunctional protein (Rep) involved in replication. A truncated form of this gene, capable of expressing the N-terminal 210 amino acids (aa) of the Rep protein, was cloned under the control of the CaMV 35S promoter and introduced into Nicotiana benthamiana using Agrobacterium tumefaciens. The same sequence was also cloned in antisense orientation. When self-pollinated progeny of 19 primary transformants were tested for resistance to TYLCV by agroinoculation, some plants proved to be resistant, particularly in the sense lines. Two such lines were further studied. The presence of the transgene was verified and its expression was followed at intervals. All plants that were resistant to TYLCV at 4 weeks postinoculation (wpi) contained detectable amounts of transgenic mRNA and protein at the time of infection. Resistance was overcome in a few plants at 9 wpi, and in most at 15 wpi. Infection of leaf discs derived from transgenic plants showed that expression of the transgene correlated with a substantial reduction of viral DNA replication. Cotransfections of tobacco protoplasts demonstrated that inhibition of viral DNA replication requires expression of the truncated Rep protein and suggested that the small ORF C4, also present in our construct, plays no role in the resistance observed. The results obtained using both transient and stable gene expression systems show that the expression of the N-terminal 210 aa of the TYLCV Rep protein efficiently interferes with virus infection.