The hrpZ Gene of Pseudomonas syringae pv. phaseolicola Enhances Resistance to Rhizomania Disease in Transgenic Nicotiana benthamiana and Sugar Beet

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.

Plant Aquaporin AtPIP1;4 Links Apoplastic H2O2 Induction to Disease Immunity Pathways

“…While an effector executes its virulence role by inhibiting H 2 O 2 production and the MAPK cascade (Zhang et al, 2007), this PTI inhibitory effect may be impaired by a PAMP. For example, when HrpZ1 is directed to the apoplast in transgenic plants (Pavli et al, 2011) or infiltrated into the apoplastic spaces of normal plants (Kvitko et al, 2007), it recognizes a PM lipid sensor (Lee et al, 2001;Haapalainen et al, 2011) and induces immune responses, including H 2 O 2 production (Dayakar et al, 2003). Under different conditions, harpin-induced immunity is attributable to SAR (Strobel et al, 1996;Dong et al, 1999Peng et al, 2004 or PTI (Lee et al, 2001;Fu et al, 2014) activated following the cytoplasmic import of apoplastic H 2 O 2 (Sang et al, 2012).…”

High Level Resistance against Rhizomania Disease by Simultaneously Integrating Two Distinct Defense Mechanisms

“…Transgene integration and absence of Ti plasmid-originated sequences was examined by a multiplex PCR assay, using specific primers to amplify the 995 bp, 459 bp and 590 bp fragments of hrpZ Psph [20], the BNYVV-derived transgene [23] and virG of A. tumefaciens [20]. Transgene expression was verified by RT-PCR using abovementioned primers.…”

“…Transgene expression was verified by RT-PCR using abovementioned primers. Plants that were PCR-positive for transgene integration and expression were subsequently examined for the accumulation of SP/HrpZ Psph by immunoblot blot analysis as previously described [20]. Plants that met all above requirements were selfed and thereof progeny seeds (T1, T2) were selected against hygromycin and kanamycin.…”

“…In terms of generating high level disease resistance, the potential of various genetic engineering approaches has been further explored. Although successful at varying levels, all such methodologies have to date exploited the introgression of a single transgene with the perspective of engineering resistance based on either the expression of gene products of pathogen [18] or non-pathogen origin [19], [20] or the RNA-silencing signaling route [21]–[23].…”

PAMP Signaling in Plant Innate Immunity

“…Transgenic Nicotiana benthamiana and sugar beet plants expressing hrpG gene of P. syringae pv. phaseolicola triggered the activation of several defense signaling genes (Pavli et al 2011 ). The HrpN of E .…”