Stepwise artificial evolution of a plant disease resistance gene

Abstract

Genes encoding plant nucleotide-binding leucine-rich repeat (NB-LRR) proteins confer dominant resistance to diverse pathogens. The wild-type potato NB-LRR protein Rx confers resistance against a single strain of potato virus X (PVX), whereas LRR mutants protect against both a second PVX strain and the distantly related poplar mosaic virus (PopMV). In one of the Rx mutants there was a cost to the broad-spectrum resistance because the response to PopMV was transformed from a mild disease on plants carrying wild-type Rx to a trailing necrosis that killed the plant. To explore the use of secondary mutagenesis to eliminate this cost of broad-spectrum resistance, we performed random mutagenesis of the N-terminal domains of this broad-recognition version of Rx and isolated four mutants with a stronger response against the PopMV coat protein due to enhanced activation sensitivity. These mutations are located close to the nucleotide-binding pocket, a highly conserved structure that likely controls the "switch" between active and inactive NB-LRR conformations. Stable transgenic plants expressing one of these versions of Rx are resistant to the strains of PVX and the PopMV that previously caused trailing necrosis. We conclude from this work that artificial evolution of NB-LRR disease resistance genes in crops can be enhanced by modification of both activation and recognition phases, to both accentuate the positive and eliminate the negative aspects of disease resistance.

Keywords: NLR; arms race; genetically modified; plant defense; plant immunity.

Fig. 1.

N-terminal random mutagenesis of RxM1 and identification of candidates. (A) Schematic of RxM1. Red, N-terminal domains; green, C-terminal LRR domain. Arrows depict error-prone PCR target region. (B) Transient expression in leaf segments of N. tabacum. PopMV-CP is coexpressed with RxM1 and the RxM1 variants identified in the screen, RxS1*M1, RxS2*M1, RxS3*M1, and RxS4M1. Photos were taken 5 d postinoculation (dpi). (Scale bar, 1 cm.) (C) Quantification of necrosis after transient coexpression of Rx variants with viral CP elicitors in N. tabacum (5 dpi) by chlorophyll content assay. A low level of chlorophyll indicates high levels of necrosis. GFP and RxM123 are used as negative and positive controls, respectively. Each bar represents an average of five biological replicates. Error bars represent 95% confidence intervals.

Fig. 2.

Amino acid changes cluster around the nucleotide-binding pocket. (A) Schematic of RxM1 variants identified from the screen, with error-prone PCR–induced amino acid changes. Responsible amino acid changes are encapsulated in red boxes. (B) Homology model of NB-ARC1-ARC2 domains of Rx (20). Red, positions of the responsible amino acid changes identified; blue, previously reported residues involved in nucleotide binding. K176 in the P loop (NB); P332 in the GxP motif (ARC1); H549 in the MHD motif (ARC2). Yellow, ADP nucleotide. The image was generated by PyMOL software.

Fig. 3.

S mutations sensitize the response. (A) Responsible amino acid change constructs S1, S2, S3, and S4, with or without the M1 mutation in the LRR, coexpressed with PopMV-CP or (B) S1, S2, S3, and S4 without the M1 mutation coexpressed with PVX-CP_KR and quantified by chlorophyll content assay 5 dpi in N. tabacum. Rx (light gray bar) and Rx coexpressed with PVX-CP_TK (dark gray bar) are used as negative and positive controls for necrosis, respectively. Each bar represents an average of five biological replicates. Error bars represent 95% confidence intervals.

Fig. 4.

Combining the S mutations causes autoactivity and requires the P loop. (A) Schematic showing the combined amino acid change constructs (S1–4) in the M1 (Upper) and wild-type (Lower) LRR backgrounds. (B) Transient expression of constructs encoding S1234 induces elicitor-independent necrosis in the M1 and wild-type LRR backgrounds. Corresponding constructs (RxM1 and Rx) with no S mutations in the N-terminal region are not autoactive. (Scale bars, 0.5 cm.) Pictures were taken 5 dpi. (C) Chlorophyll content assay to quantify necrosis after transient expression of the autoactive RxS1234 constructs containing mutations in the GKT motif 5 dpi in N. tabacum. Rx and RxS1234 are used as negative and positive controls for autoactivity, respectively. Each bar represents an average of five biological replicates. Error bars represent 95% confidence intervals. (D) Western blot for accumulation of the RxS1234 GKT mutants after transient expression in N. tabacum. RxS1234 does not show a clear band, as the tissue was extensively necrotic when collected at 3 dpi. RuBisCo is used as a loading control.

Fig. 5.

Transgenic plants carrying RxS1*M1 are resistant to PopMV. (A) Representative plants 6 wk after inoculation with PopMV. Wild-type and Rx plants show mild stunting and mosaic symptoms; RxM1 plants display the lethal trailing necrosis phenotype; and RxS1*M1 plants do not present viral symptoms. The RxM123 transgenic line is used as a positive control for PopMV resistance (27). (B) RT-PCR of cDNA from noninoculated leaf tissue harvested 3 wk postinoculation from eight different RxS1*M1 plants. For each plant, the same cDNA was used in three RT-PCR reactions to amplify PopMV coat protein, RxS1*M1 transgene, or the housekeeping gene GAPDH. Because RxS1*M1 plants are in the T1 generation, the transgene is segregating, providing an additional blinding control in the experiment (uninfected RxS1*M1 T1 plants are phenotypically identical). These results were repeated on at least three separate occasions.