Heterologous Expression of the AtNPR1 Gene in Olive and Its Effects on Fungal Tolerance

Abstract

The NPR1 gene encodes a key component of systemic acquired resistance (SAR) signaling mediated by salicylic acid (SA). Overexpression of NPR1 confers resistance to biotrophic and hemibiotrophic fungi in several plant species. The NPR1 gene has also been shown to be involved in the crosstalk between SAR signaling and the jasmonic acid-ethylene (JA/Et) pathway, which is involved in the response to necrotrophic fungi. The aim of this research was to generate transgenic olive plants expressing the NPR1 gene from Arabidopsis thaliana to evaluate their differential response to the hemibiotrophic fungus Verticillium dahliae and the necrotroph Rosellinia necatrix. Three transgenic lines expressing the AtNPR1 gene under the control of the constitutive promoter CaMV35S were obtained using an embryogenic line derived from a seed of cv. Picual. After maturation and germination of the transgenic somatic embryos, the plants were micropropagated and acclimated to ex vitro conditions. The level of AtNPR1 expression in the transgenic materials varied greatly among the different lines and was higher in the NPR1-780 line. The expression of AtNPR1 did not alter the growth of transgenic plants either in vitro or in the greenhouse. Different levels of transgene expression also did not affect basal endochitinase activity in the leaves, which was similar to that of control plants. Response to the hemibiotrophic pathogen V. dahliae varied with pathotype. All plants died by 50 days after inoculation with defoliating (D) pathotype V-138, but the response to non-defoliating (ND) strains differed by race: following inoculation with the V-1242 strain (ND, race 2), symptoms appeared after 44-55 days, with line NPR1-780 showing the lowest disease severity index. This line also showed good performance when inoculated with the V-1558 strain (ND, race 1), although the differences from the control were not statistically significant. In response to the necrotroph R. necatrix, all the transgenic lines showed a slight delay in disease development, with mean area under the disease progress curve (AUDPC) values 7-15% lower than that of the control.

Keywords: Olea europaea; SAR response; Verticillium wilt; genetic transformation; soil-borne pathogens; white root rot.

FIGURE 1

Obtainment of olive lines transformed with the NPR1 gene from Arabidopsis thaliana. (A) Globular somatic embryos used for inoculation with A. tumefaciens. (B) Transgenic callus from the NPR1-814 line growing in ECO selection medium supplemented with 150 mg/L paromomycin. (C) Transgenic callus from the NPR1-814 line after 3 weeks of selection in liquid ECO medium supplemented with 25 mg/L paromomycin. (D) Transgenic somatic embryos cultured on maturation medium over a semi-permeable cellulose acetate membrane. (E) Micropropagated shoots from the NPR1-814 line cultured on RP medium. (F) Transgenic NPR1-814 shoots cultured on RP medium supplemented with activated charcoal after 3 days in liquid medium with 10 mg/L IBA for rooting. (G) Acclimated plants from the NPR1-224 line after 6 weeks in the growth chamber. (H) From left to right, acclimated plants derived from the transgenic NPR1-224, NPR1-780, and NPR1-814 lines and the non-transformed P1 line after 9 months of growth in the greenhouse. Bars correspond to 0.5 cm (A,B,D,E), 1 cm (F,G), and 5 cm (H).

FIGURE 2

PCR amplifications of the nptII (A) and AtNPR1 (B) gene fragments from genomic DNA extracted from embryogenic callus from the different transgenic AtNPR1 lines and non-transformed line P1.780, 814, and 224: transgenic AtNPR1 lines; P1, non-transgenic control; C–, negative control (without DNA); C+, 35S, AtNPR1 binary plasmid; M, molecular marker.

FIGURE 3

Relative AtNRP1 expression in leaves from transgenic olive plants. The mRNA values are shown relative to the lowest value obtained in line NPR1-814, which was assigned a value of 1. Data correspond to the mean ± SD of three independent RNA extractions.

FIGURE 4

Western blot detection of AtNPR1 protein in leaf extracts of control (C) and transgenic olive plants. Plants were sprayed with 0.5 mM salicylic acid until runoff, and leaf samples were obtained before (–SA) and 24 h after SA treatment (+SA).

FIGURE 5

Expression of the PR1-like gene XM_022999257.1 in the leaves of transgenic olive plants. Gene expression is shown as relative to the control non-transgenic plant value, which was given an arbitrary value of 1. Asterisks indicate significant differences from the control by t-test at P = 0.05.

FIGURE 6

Chitinase activity in the leaves of control (P1) and transgenic olive plants expressing the AtNPR1 gene. The data correspond to the mean ± SD of three independent protein extractions.

FIGURE 7

Average values of area under the disease progress curve (AUDPC) in control (P1) and transgenic AtNPR1 olive plants inoculated with Verticillium dahliae, non-defoliating strains V-1242 and V-1558. Data represent the mean ± SE. Asterisks indicate significant differences from control P1 by the Mann–Whitney U test at P = 0.05.

FIGURE 8

Average values of area under the disease progress curve (AUDPC) in control (P1) and transgenic AtNPR1 olive plants inoculated with Rosellinia necatrix. Data represent the mean ± SE. Different letters indicate significant differences by LSD test at P = 0.05.