Detection of the virulent form of AVR3a from Phytophthora infestans following artificial evolution of potato resistance gene R3a

Abstract

Engineering resistance genes to gain effector recognition is emerging as an important step in attaining broad, durable resistance. We engineered potato resistance gene R3a to gain recognition of the virulent AVR3aEM effector form of Phytophthora infestans. Random mutagenesis, gene shuffling and site-directed mutagenesis of R3a were conducted to produce R3a* variants with gain of recognition towards AVR3aEM. Programmed cell death following gain of recognition was enhanced in iterative rounds of artificial evolution and neared levels observed for recognition of AVR3aKI by R3a. We demonstrated that R3a*-mediated recognition responses, like for R3a, are dependent on SGT1 and HSP90. In addition, this gain of response is associated with re-localisation of R3a* variants from the cytoplasm to late endosomes when co-expressed with either AVR3aKI or AVR3aEM a mechanism that was previously only seen for R3a upon co-infiltration with AVR3aKI. Similarly, AVR3aEM specifically re-localised to the same vesicles upon recognition by R3a* variants, but not with R3a. R3a and R3a* provide resistance to P. infestans isolates expressing AVR3aKI but not those homozygous for AVR3aEM.

Figure 1. Four rounds of mutagenesis and shuffling identified R3a mutants with enhanced recognition of AVR3a^EM and disease responses (R3a*).

(a) Schematic showing locations of non-synonymous mutations found in the LRRs of R3a* clones isolated from the four rounds of mutagenesis and shuffling (Rd1 to Rd4). LRRs containing amino acids under diversifying selection are shaded above. (b) Representative N. benthamiana leaf showing responses of best performing clones from second, third and fourth rounds (Rd2-1, Rd3-1 & Rd4-1) to AVR3a^EM (EM), compared to responses of wild-type R3a to AVR3a^KI (KI) and AVR3a^EM five days after co-infiltration with resistance genes under transcriptional control of R3a promoter. (c) Mean disease scores from the four experiments, each of nine days duration, for different infiltration mixtures in upper (hatched) and lower (solid) paired leaves. Error bars show +/− standard error. (d) Time-course of percentage of sites showing necrosis development, greater than 50% necrosis of individual infiltrated sites, for the five infiltrated mixtures. Mean percentages of the four experiments. Each experiment includes data for 40 infiltration sites (upper and lower leaves combined) and error bars show +/− standard errors.

Figure 2. HR responses resulting from R3a* recognition of AVR3a^EM and AVR3a^KI, like those caused by wild-type R3a recognition of AVR3a^KI, are dependent on SGT1 and HSP90.

SGT1- and HSP90-silenced plants were produced using TRV-based vectors. These plants and control plants inoculated with TRV:eGFP were infiltrated with different combinations of Agrobacterium cultures designed to express R3a, R3a* variants, AVR3a^KI (KI) or AVR3a^EM (EM). The percentage of sites (N = 12) showing HR responses six days after infiltration was recorded. The graph shows the mean percentages from three independent experiments with the exception that the dependence on HSP90 of Rd4-1 responses was only tested in a single experiment. The non-host bacterial pathogen Erwinia amylovora was used as a control for an SGT1- and HSP90-independnet HR response. Error bars show +/− standard error. Zero values have been transformed to 1% to facilitate their observation.

Figure 3. YFP fusions to R3a* variants re-localize to vesicles after the perception of both of AVR3a^KI and AVR3a^EM, whereas YFP-R3a remains cytoplasmic in the presence of AVR3a^EM.

Two days after infiltration of mixtures of Agrobacterium cultures designed to express AVR3a^KI, AVR3a^EM, YFP-R3a or YFP fusions to the R3a* variants, infiltrated N. benthamiana leaf tissue was examined under a confocal laser scanning microscope. Scale bar  = 50 µm.

Figure 4. Both YC-AVR3a^KI and YC-AVR3a^EM when co-expressed with YN-R3a* fusions give vesicle associated YFP fluorescence like YC-AVR3a^KI and YN-R3a, whereas YC-AVR3a^EM and YN-R3a do not.

Two days after infiltration of mixtures of Agrobacterium cultures designed to express YC-AVR3a^KI, YC-AVR3^EM, YN-R3a or YN fusions to the R3a* variants, infiltrated N. benthamiana leaf tissue was examined under a confocal laser scanning microscope. Representative images from two experiments. Scale bar  = 50 µm.

Figure 5. R3a and R3a* variants expressed from Agrobacterium reduce the spread of a P. infestans strain expressing AVR3a^KI, but not the spread of a strain expressing only AVR3a^EM.

(a) Means of lesion diameters measured 12 days after drop inoculation of agro-infiltrated areas with strain 7804.b (KI/KI). (b) Means of lesion diameters measured 8 days after drop inoculation of agro-infiltrated areas with strain 88069 (EM/EM). (a) and (b) show representative experiments from sets of three and five repeated experiments, respectively. For both (a) and (b), error bars show +/− standard errors, N = 30. EVC indicates empty vector control.

Figure 6. R3a and R3a* variants expressed via the R3a promoter in transgenic plants protect the susceptible cultivar Ranger Russet from P. infestans strain P6752, which is heterozygous for AVR3a^KI and AVR3a^EM, but not from strain US-8 BF-6, which is homozygous for AVR3a^EM.

Non-transgenic plants were used as a control for susceptibility. Transgenic plants expressing R3a or Rpi-vnt1 were used as positive controls for resistance to P6752 or US-8 BF-6, respectively. Representative plants were photographed at 11dpi.