Sequence Exchange between Homologous NB-LRR Genes Converts Virus Resistance into Nematode Resistance, and Vice Versa

Abstract

Plants have evolved a limited repertoire of NB-LRR disease resistance (R) genes to protect themselves against myriad pathogens. This limitation is thought to be counterbalanced by the rapid evolution of NB-LRR proteins, as only a few sequence changes have been shown to be sufficient to alter resistance specificities toward novel strains of a pathogen. However, little is known about the flexibility of NB-LRR R genes to switch resistance specificities between phylogenetically unrelated pathogens. To investigate this, we created domain swaps between the close homologs Gpa2 and Rx1, which confer resistance in potato (Solanum tuberosum) to the cyst nematode Globodera pallida and Potato virus X, respectively. The genetic fusion of the CC-NB-ARC of Gpa2 with the LRR of Rx1 (Gpa2_CN/Rx1_L) results in autoactivity, but lowering the protein levels restored its specific activation response, including extreme resistance to Potato virus X in potato shoots. The reciprocal chimera (Rx1_CN/Gpa2_L) shows a loss-of-function phenotype, but exchange of the first three LRRs of Gpa2 by the corresponding region of Rx1 was sufficient to regain a wild-type resistance response to G. pallida in the roots. These data demonstrate that exchanging the recognition moiety in the LRR is sufficient to convert extreme virus resistance in the leaves into mild nematode resistance in the roots, and vice versa. In addition, we show that the CC-NB-ARC can operate independently of the recognition specificities defined by the LRR domain, either aboveground or belowground. These data show the versatility of NB-LRR genes to generate resistance to unrelated pathogens with completely different lifestyles and routes of invasion.

Figure 1.

The chimera Gpa2_CN/Rx1_L confers a constitutive cell death response. A, The domain-swap construct Gpa2_CN/Rx1_L was obtained by exchanging the LRR domain of Gpa2 with the corresponding domain of Rx1 using the ApalI restriction site (after Slootweg et al., 2013). B, Agroinfiltration of the chimera Gpa2_CN/Rx1_L in leaves of N. benthamiana results in a constitutive cell death response when expressed under the control of the native RXI promoter. For the wild-type Rx1 gene, a specific cell death response was obtained in the presence of the avirulent PVX elicitor CP106 when expression was driven by either the CaMV 35S promoter or its native promoter. The virulent PVX elicitor CP105 and YFP were used as controls.

Figure 2.

Restoring PVX resistance in potato by reducing Gpa2_CN/Rx1_L expression levels. A, A second translation initiation site was introduced in the construct 35S_LS::Gpa2_CN/Rx1_L to obtain leaky scanning of ribosomes (Kozak, 1995, 1999) and a subsequent reduction of the protein expression levels (Slootweg et al., 2010). B, Agroinfiltration of N. benthamiana leaves with 35S::Gpa2_CN/Rx1_L results in a constitutive cell death response in the absence of the PVX elicitor, whereas no such autoactivation response was observed for 35S_LS::Gpa2_CN/Rx1_L. The activation of a specific cell death response was detected for 35S_LS::Gpa2_CN/Rx1_L in the presence of the avirulent PVX elicitor CP106, similar to the wild-type Rx1 gene under the control of the CaMV 35S promoter. The virulent PVX elicitor CP105 and GFP were used as controls. C, Quantification of cell death by measuring the chlorophyll contents of cells upon agroinfiltration of the constructs in N. benthamiana (after Harris et al., 2013) to confirm the differences observed by visual scoring. Cell death results in lower absorption values at A₆₅₅ due to chlorophyll loss (days post inoculation = 2; n = 8; mean ± se). D, A greenhouse virus resistance assay was performed on transgenic potato plants expressing the wild-type Rx1 gene under the control of the CaMV 35S promoter and the domain-swap construct Gpa2_CN/Rx1_L under the control of the leaky scanning 35S_LS promoter. The diploid potato clone SH, which contains the endogenous Rx1 gene, was used as the resistant control, and the diploid potato clone line V, which was used for the transformation of the constructs, was used as a susceptible control. Leaf material was collected from secondary infected leaves of the plant apex 3 weeks after infection with the avirulent strain PVX_UK3 or the virulent strain PVX_HB, and systemic spreading of the virus in the plants was detected by DAS-ELISA (mean ± sd).

Figure 3.

Restoring nematode resistance to G. pallida in potato by the chimera R13rggG5. A, Schematic overview of the domain-swap constructs used in this study to reconstruct a functional chimera by combining Gpa2 LRR segments required for RBP-1 recognition and Rx1 LRR segments compatible with the Rx1 CC-NB-ARC response domains. The top row shows sequence fragments in the LRR domain between Gpa2 (G4) and Rx1 (R4), with the positions of the break points in the amino acid sequence given above. If the numbering of the positions differs between Rx1 and Gpa2, both numbers are given. The second row shows the domain architecture with the coiled coil (CC = 1), nucleotide-binding (NB = 2), and ARC1 and ARC2 (=3) subdomains and the LRR domain (=4) followed by an acidic tail (=5). In the third row, the amino acid positions polymorphic between Rx1 and Gpa2 are indicated. The Rx1 sequence is depicted in black and the Gpa2 sequence in white in the chimeric constructs. Specific combinations of LRR segments derived from Gpa2 or Rx1 are indicated in lowercase letters in the construct names, whereas complete domains are indicated in uppercase letters (after Slootweg et al., 2013). B, Functional analysis of the six domain-swap constructs in the presence of the activating RBP-1 variant Rook6 and the nonactivating RBP-1 variant Rook4 upon agroinfiltration in leaves of N. benthamiana (after Sacco et al., 2009). The Gpa2 gene was used as a control. Expression of the constructs was driven by the CaMV 35S promoter. C, Quantification of the cell death response induced by the six domain-swap constructs in the presence of the nematode effector RBP-1 variant Rook6 based on a reduction in chlorophyll content (after Harris et al., 2013). GFP was used as a negative control. Multiple infiltration spots were collected per construct and pooled for two different plants (n = 6; mean ± se). D, In planta protein expression of the two chimeric proteins R13ggrG5 and R13gggR5, which showed a loss-of-function phenotype, was confirmed by agroinfiltration of the constructs in leaves of N. benthamiana followed by western blotting using an anti-Myc antibody. A diluted sample of GFP-Myc was included as a control, and Commassie Brilliant Blue (CBB) staining of the gel was used to check the loading of equal amounts of protein. E, Greenhouse nematode resistance assay on transgenic potato lines harboring the chimeric constructs R13G45 (also known as Rx1_CN/Gpa2_L; see Supplemental Fig. S1), R13grgG5, R13ggrG5, R13G4G5, and R13rggG5 under the control of the CaMV 35S promoter. Transgenic plants harboring the full-length Gpa2 gene were used as resistant controls (line 9.2), like the resistant potato clone SH, which harbors the Gpa2 gene. A transgenic line containing the empty vector (EV) was used as a susceptible control. Roots were inoculated with the avirulent Pa₂-D383 population of the potato cyst nematode G. pallida. Two to four independent transgenic lines were assayed in multiple replicates for each transgene. Cysts were counted on these plants at 16 weeks post inoculation, and average numbers ± sd are shown.

Figure 4.

The Rx1 and Gpa2 promoters are exchangeable, driving both nematode resistance in potato roots as well as PVX resistance in potato shoots. A, A specific cell death phenotype was obtained for Rx1 on leaves of N. benthamiana when coexpressed with the avirulent elicitor CP106. The virulent elicitor CP105 and YFP were used as negative controls. Rx1 was expressed either from the endogenous RXI or the GPAII promoter. Similar results were obtained for Gpa2 when expressed under the control of the RXI promoter and the GPAII promoter in the presence of the activating RBP-1 variant Rook6. The nonactivating RBP-1 variant Rook4 and GUS were used as negative controls. B, Greenhouse virus resistance assay. The mean absorbance values (A₄₀₅) are shown for homogenate derived from secondary compound leaves in ELISA of transgenic potato plants. Rx1 was expressed from its own promoter (RXI) or the Gpa2 promoter (GPAII). Transgenic lines harboring the genomic Rx1 bacterial artificial chromosome (BAC) sequence were used as positive controls. Leaves were harvested 3 weeks after primary leaf inoculation with the avirulent strain PVX_UK3 or the virulent strain PVX_HB. Between four and 12 plants from two to four independent lines were assayed per construct (mean ± sd). C, Greenhouse nematode resistance assay on transgenic potato plants harboring the Gpa2 gene under the control of either the endogenous RXI or GPAII promoter and terminator regions. Plants were tested with the avirulent Pa₂-D383 population and the virulent population Pa₃-Rookmaker of the potato cyst nematode G. pallida. Three independent transgenic lines were assayed in multiple replicates for each transgene. Cysts were counted on these plants at 16 weeks post inoculation, and average numbers ± sd are shown.