Fig. 3: AvrPm4⁹⁶²²⁴ interacts with both Pm4b isoforms V1 and V2.

a,b, Split-luciferase assay showing the interaction of NLUC-tagged Pm4b-V1 (a) and NLUC-tagged Pm4b-V2 (b) with CLUC-tagged AvrPm4⁹⁶²²⁴ and CLUC-tagged avrPm4^4AB-2 in N. benthamiana. Co-expression of the two Pm4b isoforms with BgtE-5764 and of AvrPm4⁹⁶²²⁴ with WTK4 served as negative controls. AvrPm4⁹⁶²²⁴ interacts with both Pm4b isoforms. Each treatment was assessed with two technical and six biological replicates, and the experiments were repeated three times (total n = 18 biological replicates). In each box plot, the median is shown as a central line in a box that spans from the 25th to the 75th percentile. The inner whiskers show the 95% confidence interval of the median, and the outer whiskers show the most extreme data points within 1.5 times the interquartile range. Significance and P values (significance threshold of P < 0.05) were calculated with a two-sided ANOVA followed by multiple comparisons of means (contrast analyses) with Bonferroni correction (***P ≤ 0.001; **P ≤ 0.01; *P ≤ 0.05; NS, not significant). c,d, Subcellular colocalization in N. benthamiana of mTurquoise–AvrPm4⁹⁶²²⁴ (cyan), Pm4b-V1 (magenta) and Pm4b-V2 (yellow). When co-expressed with Pm4b-V1, AvrPm4⁹⁶²²⁴ colocalizes to the cytoplasm (c; scale bars, 10 μm), while co-expression with Pm4b-V2 redirects AvrPm4 to the ER (d; scale bars, 5 μm). The experiment was repeated twice. e, In vitro kinase assay showing autophosphorylation and transphosphorylation activity of purified Pm4 variants in the presence of [ɣ-³²P]ATP. All constructs shown have an MBP tag. All reactions were supplemented with the common substrate MyBP (21 kDa, indicated by triangles). Constructs included Pm4b-V1, Pm4b-V2ΔTMD and their kinase-dead mutants (D170N and D188N), as well as a truncated construct containing only the kinase domain (Pm4b-Exon1–5; calculated protein size, 77.5 kDa). His-MBP alone served as a negative control. Wild-type Pm4b-V1 (calculated protein size, 106 kDa) and Pm4b-V2ΔTMD (calculated protein size, 107 kDa) were found to migrate at a size of around 120 kDa (see f and Extended Data Fig. 5a for better resolution). Wild-type Pm4b-V1 and Pm4b-V2ΔTMD showed kinase activity (indicated by a single asterisk). Truncated proteins migrating at around 90 kDa (indicated by a double asterisk) were also detected. The substitutions D170N and D188N reduced or abolished kinase activity, respectively. Coomassie-stained proteins on polyvinylidene difluoride (PVDF) membranes served as loading controls. CBB, Coomassie brilliant blue. f, In vitro phosphorylation assay of AvrPm4⁹⁶²²⁴ and avrPm4^4AB-2 by Pm4. All constructs shown have an MBP tag. Pm4b-V1 and Pm4b-V2ΔTMD were incubated with AvrPm4⁹⁶²²⁴ and avrPm4^4AB-2 in the presence of [ɣ-³²P]ATP. AvrPm4⁹⁶²²⁴ (110 kDa, with truncated products ranging between 70 kDa and 100 kDa (Extended Data Fig. 5b), indicated by two single squares) and avrPm4^4AB-2 (indicated by a circle) were phosphorylated by both Pm4b isoforms. As a control, the same AvrPm4 constructs were incubated with kinase-dead Pm4b-V1(D188N). The kinase assays were repeated twice.

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