Transformation of sweet orange [Citrus sinensis (L.) Osbeck] with pthA-nls for acquiring resistance to citrus canker disease

The COOH terminal of pthA encoding three nuclear localizing signals (NLS) was amplified by polymerase chain reaction (PCR) from the plasmid of Xanthomonas axonopodis pv. citri, the pathogen of citrus canker disease. Then the sense and antisense strands of the nls were cloned into pBI121 vector. pthA-nls driven by the CaMV35 s promoter was transferred into sweet orange via Agrobacterium -mediated transformation. Successful integration was confirmed by PCR and Southern blotting, and 12 sense-nls (nls +) and 9 antisense-nls (nls −) transgenic clones were obtained. The expression of nls fragment was analyzed by RT–PCR, Real time q-PCR and Western blotting, in which the specific NLS protein was detected only in nls + transgenic clones. In an in vitro assay, when pin-puncture inoculation was performed with 2.5 × 107 cfu/ml of bacterial solution, the nls + transgenic clones showed no typical lesion development, while typical symptoms were observed in the wild types and the nls − transgenic clones. In vivo assay results indicated that the nls + transgenic clones showed less disease incidence, in comparison with the wild types and the nls − transgenic clones, when pin-puncture inoculation was performed with 104–105 cfu/ml. The minimum disease incidence was 23.3% for ‘Sucarri’ sweet orange and 33.3% for ‘Bingtang’ sweet orange. When 104–107 cfu/ml of pathogen was spray inoculated, the nls + transgenic clones did not show any symptom, and even the concentration raised to 109 cfu/ml, the disease incidence was 20–80%, while the wild types and the nls − transgenic clones had 100% disease development with whatever concentration of inoculum. Two transgenic clones were confirmed to be resistant to citrus canker disease in the repeated inoculation. The results suggested that the transformation of nls sense strands may offer an effective way to acquire resistance to citrus canker disease.