Inducible expression of p50 from TMV for increased resistance to bacterial crown gall disease in tobacco

Abstract

The dominant tobacco mosaic virus (TMV) resistance gene N induces a hypersensitive response upon TMV infection and protects tobacco against systemic spread of the virus. It has been proposed to change disease resistance specificity by reprogramming the expression of resistance genes or their corresponding avirulence genes. To reprogramme the resistance response of N towards bacterial pathogens, the helicase domain (p50) of the TMV replicase, the avirulence gene of N, was linked to synthetic promoters 4D and 2S2D harbouring elicitor-responsive cis-elements. These promoter::p50 constructs induce local necrotic lesions on NN tobacco plants in an Agrobacterium tumefaciens infiltration assay. A tobacco genotype void of N (nn) was transformed with the promoter::p50 constructs and subsequently crossed to NN plants. Nn F1 offspring selected for the T-DNA develop normally under sterile conditions. After transfer to soil, some of the F1 plants expressing the 2S2D::p50 constructs develop spontaneous necrosis. Transgenic Nn F1 plants with 4D::p50 and 2S2D::p50 expressing constructs upregulate p50 transcription and induce local necrotic lesions in an A. tumefaciens infiltration assay. When leaves and stems of Nn F1 offspring harbouring promoter::p50 constructs are infected with oncogenic A. tumefaciens C58, transgenic lines harbouring the 2S2D::p50 construct induce necrosis and completely lack tumor development. These results demonstrate a successful reprogramming of the viral N gene response against bacterial crown gall disease and highlight the importance of achieving tight regulation of avirulence gene expression and the control of necrosis in the presence of the corresponding resistance gene.

Resistance analysis of cherry rootstock ‘CDR-1’ (Prunus mahaleb) to crown gall disease

“…Plants are often exposed to many various bacterial, viral, and fungal pathogens but have evolved potent defense systems to protect themselves [7]. In defense responses of plants, the identification of microbial pathogens plays a key role, as it "turns on" the signal transduction pathway which activates the expression of numerous pathogenresponsive genes [8,9]. These disease resistance genes are crucial for identifying the effector proteins during the process of pathogen infection [7].…”

“…Therefore, to obtain plants that are resistant to crown gall disease, much research has been devoted to producing sense and antisense strands of the oncogene sequence by placing these sequences between opposing strong constitutive promoters [13], or to silencing the involved bacterial oncogenes by using premature stop codons [14]. The study of Niemeyer et al (2014) demonstrated a successful reprogramming of the viral N gene response against crown gall disease [9]. In recent years, Rosalia Deeken's group has been working on the molecular mechanism between crown gall disease and A. tumefaciens in Arabidopsis thaliana [8,[15][16][17][18].…”

Functional dissection of a strong and specific microbe‐associated molecular pattern‐responsive synthetic promoter

“…For example, avirulence genes can be linked with pathogen-responsive promoters to reprogramme corresponding resistance genes to the specificity of the promoter. Recently, the TMV resistance gene N was reprogrammed to protect tobacco against Agrobacterium-mediated crown gall disease (Niemeyer et al, 2014). In this system, unspecific expression of the avirulence gene also led to background necrosis and to spreading necrosis after Agrobacterium infection.…”

Synthetic promoters in planta

“…Recently, two efficient synthetic promoters (4D and 2S2D) were constructed by coupling either the D or the D/S elements from parsley plants to the CaMV 35S core promoter (Niemeyer et al 2014). Both promoters were fused to the helicase domain (p50) of TMV replicase and cloned into a T-DNA vector.…”