Generation of broad-spectrum disease resistance by overexpression of an essential regulatory gene in systemic acquired resistance

Abstract

The recently cloned NPR1 gene of Arabidopsis thaliana is a key regulator of acquired resistance responses. Upon induction, NPR1 expression is elevated and the NPR1 protein is activated, in turn inducing expression of a battery of downstream pathogenesis-related genes. In this study, we found that NPR1 confers resistance to the pathogens Pseudomonas syringae and Peronospora parasitica in a dosage-dependent fashion. Overexpression of NPR1 leads to enhanced resistance with no obvious detrimental effect on the plants. Thus, for the first time, a single gene is shown to be a workable target for genetic engineering of nonspecific resistance in plants.

Figure 1

Analysis of PR1 expression, NPR1 protein and mRNA in wild-type and NPR1 cDNA transgenic plants. (A) Comparison of INA-induced PR1 expression (as determined by Northern blot analysis), uninduced NPR1 protein (as measured by ELISA) and mRNA levels in NPR1 cDNA transgenic plants (as determined by Northern blot analysis) to those in wild type. All the analyses were performed twice with similar results. The transgenic plants were classified into three groups, designated as 35S-NPR1-L (low), 35S-NPR1-M (medium), and 35S-NPR1-H (high), respectively, based on the levels of induced PR1 expression. (B) Immunoblot showing the different levels of NPR1 protein in wild-type and NPR1 cDNA transgenic plants.

Figure 2

Analysis of disease resistance to the bacterial pathogen Pseudomonas syringae pv. maculicola ES4326 in wild-type and NPR1 cDNA transgenic plants. (A) Growth of Psm ES4326 in wild-type and NPR1 cDNA transgenic plants. The leaves of 4-week-old wild-type and the transgenic plants were inoculated with Psm ES4326 at OD₆₀₀= 0.001. At 0, 0.5, 1, 2, and 3 days after the inoculation, infected leaves were collected and the bacterial growth was determined (10). Bars = 95% confidence limits of log-transformed data (38). Eight samples were taken for each time point. cfu, Colony forming unit. (B) Disease symptoms caused by Psm ES4326 in wild-type and NPR1 cDNA transgenic plants. From left to right, the order of the leaves is: wild type, 35S-NPR1-L, 35S-NPR1-M, and 35S-NPR1-H. The photograph was taken 3 days after infection.

Figure 3

Analysis of resistance to the oomycete pathogen Peronospora parasitica strain Noco in wild-type and NPR1 cDNA transgenic plants. (A) Disease ratings of wild-type and NPR1 cDNA transgenic plants after the infection with P. parasitica Noco. Two-week-old soil-grown seedlings of wild-type and transgenic plants were sprayed to imminent runoff with spores of P. parasitica Noco (∼3 × 10⁴/ml). Six days after infection, 30 plants of each line were sampled to rate disease symptoms. Ratings were defined as follows: 0, no conidiophores on the plant; 1, no more than 5 conidiophores per infected plant; 2, 6–20 conidiophores on a few infected leaves; 3, 6–20 conidiophores on most of the infected leaves; 4, 5 or more conidiophores on all infected leaves; 5, 20 or more conidiophores on all infected leaves. The data were analyzed by using Mann–Whitney U tests (38). (B) Conidiophores observed in wild-type and NPR1 cDNA transgenic plants seven days after inoculation with P. parasitica Noco. Plants were examined under a dissecting microscope. (C) Trypan blue staining of P. parasitica-infected leaves of wild-type and NPR1 cDNA transgenic plants seven days after infection. Seedlings of wild-type and transgenic plants were stained with trypan blue and mounted for observation under a compound microscope (36).

Figure 4

Analysis of PR gene expression in wild-type and NPR1 cDNA transgenic plants after infection by Psm ES4326. At 0, 3, 6, 12, and 24 hr after inoculation with Psm ES4326 at OD₆₀₀ = 0.001, infected leaves were collected from ten individual plants and total RNA was extracted from the leaf tissue. RNA blot analyses were performed by using PR1, BGL2 (PR2), PR5, and 18S rRNA as probes (36).