Abstract
To explore possible sources of transgenic resistance to the rhizomania-causing Beet necrotic yellow vein virus (BNYVV), Nicotiana benthamiana plants were constructed to express the harpin of Pseudomonas syringae pv. phaseolicola (HrpZPsph). The HrpZ protein was expressed as an N-terminal fusion to the PR1 signal peptide (SP/HrpZ) to direct harpin accumulation to the plant apoplast. Transgene integration was verified by mPCR in all primary transformants (T0), while immunoblot analysis confirmed that the protein HrpZPsph was produced and the signal peptide was properly processed. Neither T0 plants nor selfed progeny (T1) showed macroscopically visible necrosis or any other macroscopic phenotypes. However, plants expressing the SP/HrpZPsph showed increased vigor and grew faster in comparison with non-transgenic control plants. Transgenic resistance was assessed after challenge inoculation with BNYVV on T1 progeny by scoring of disease symptoms and by DAS-ELISA at 20 and 30 dpi. Transgenic and control lines showed significant differences in terms of the number of plants that became infected, the timing of infection and the disease symptoms displayed. Plants expressing the SP/HrpZPsph developed localized leaf necrosis in the infection area and had enhanced resistance upon challenge with BNYVV. In order to evaluate the SP/HrpZ-based resistance in the sugar beet host, A. rhizogenes-mediated root transformation was exploited as a transgene expression platform. Upon BNYVV inoculation, transgenic sugar beet hairy roots showed high level of BNYVV resistance. In contrast, the aerial non-transgenic parts of the same seedlings had virus titers that were comparable to those of the seedlings that were untransformed or transformed with wild type R1000 cells. These findings indicate that the transgenically expressed SP/HrpZ protein results in enhanced rhizomania resistance both in a model plant and sugar beet, the natural host of BNYVV. Possible molecular mechanisms underlying the enhanced resistance and plant growth phenotypes observed in SP/HrpZ transgenic plants are discussed.
Highlights
Rhizomania disease of sugar beet is responsible for a very significant reduction in crop’s productivity globally, as a consequence of a considerable decrease in root yield, sugar content and juice purity [1,2]
We examined the expression of hin1, hsr203J, SIPK, WIPK, which are hypersensitive response (HR)-associated genes, and AOX, COI1, NPR1 and PR1a, which are involved in active oxygen species (AOS), jasmonic acid (JA), salicylate (SA)- and PR-dependent defense pathways, respectively [48,49,50,51,52,53]
The transgenic expression of the harpin HrpZPsph from P. syringae pv. phaseolicola, has been deployed for a first time, as a means for evaluating the ability of the protein to elicit a general defense response which would result in protection against rhizomania, a serious disease of the sugar beet crop
Summary
Rhizomania disease of sugar beet is responsible for a very significant reduction in crop’s productivity globally, as a consequence of a considerable decrease in root yield, sugar content and juice purity [1,2]. Given the absence of other control strategies, the only substantial means to ensure a viable crop production in rhizomania infested areas is the use of varieties bred for resistance to the virus [3] In this respect, coping with rhizomania to date has been based mainly on cultivars endowed with the Rz1 resistance gene (‘‘Holly’’ source), a dominant gene conferring sufficiently high levels of protection against BNYVV [4,5]. In addition to conventional breeding methodologies that led to all currently rhizomania resistant sugar beet varieties, various genetic engineering approaches have been studied for the purpose of enhancing disease resistance These include pathogen-derived resistance (PDR), relying on the transgenic expression of viral genes/sequences [6,7], antibodymediated resistance [8] and RNA silencing-mediated resistance, the most successful variant of PDR [9,10,11]. Recent changes in the field and molecular BNYVV epidemiology, as manifested by the emergence of type-A virus strains capable of compromising the Rz1-based resistance [12,13] and the spread of highly pathogenic type P-isolates [14], necessitate further research for alternative forms of resistance against BNYVV
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