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.

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

R3a and R3a* variants expressed via the <i>R3a</i> promoter in transgenic plants protect the susceptible cultivar Ranger Russet from <i>P. infestans</i> 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.

<p>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.</p

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.

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

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

<p>(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.</p

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.

<p>SGT1- and HSP90-silenced plants were produced using TRV-based vectors. These plants and control plants inoculated with TRV:<i>eGFP</i> were infiltrated with different combinations of <i>Agrobacterium</i> 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 <i>Erwinia amylovora</i> 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.</p