To investigate further the translocation of Avr3a from haustoria into the plant cell, the signal peptide (SP)-RXLR-EER-encoding domain of Avr3a, and a version in which the motifs were replaced with alanine residues (SP-AAAA-AAA) were fused to the amino terminus of the Escherichia coli gusA gene, which encodes β-glucuronidase (GUS), and stably expressed in P. infestans. GusA was chosen because its product is active within plant cells but inactive in the apoplast19, and is thus an ideal reporter for translocation of proteins to the inside of plant cells. SP-RXLR-EER–GUS and SP-AAAA-AAA–GUS transformants were selected that expressed and secreted high levels of active GUS into culture medium (Supplementary Fig. 6). However, when infecting potato, GUS activity was observed only with the SP-RXLR-EER–GUS transformants, and only within plant cells in contact with haustoria (Fig. 3 and Supplementary Fig. 7). No GUS activity was observed within plant cells or in the apoplast in the case of the SP-AAAA-AAA–GUS transformants (Fig. 3 and Supplementary Fig. 7), indicating that GUS was not translocated into the plant cell.
1/ It would be nice to see a decrease of nuclear signal as you move away from the IH/BIC plant cell. Sometimes there are several cells with stained nuclei - one would expect the signal to become weaker and weaker as the protein gets diluted. Some clear documentation of the weakening of the signal as you move away from the first infected cells would be nice.
2/ A question arose last year about whether the artificial NLS is actually carrying the effector inside the plant cells since there are papers about NLS sequences acting as cell-penetrating peptides.