Figure 1.
CRN8 domain composition and phylogenetic analysis of CRN8 paralogs.
(A) Cartoon of the three different motif combinations of CRN8 orthologs present in three sequenced Phytophthora genomes. The signal peptide (SP, black), LFLAK motif (blue), DWL motif (green), the NLS domain (red), and the kinase-homologous D2 domain (yellow) are presented as separate building blocks. The copy number of CRN8 paralogs and pseudogenes for three different sequenced Phytophthora genomes is stated alongside each CNR8 archetype. (B) Two phylogenetic trees of P. infestans (blue) and P. ramorum (orange) CRN8 paralogs based on amino acid sequence; the left side shows the N-terminus and the right side indicates the C-terminus. The originally identified CRN8 sequence from P. infestans isolate H88069 was also included (named CRN8). The other sequences are presented by their genome number. The N-terminal amino acid sequences in the left tree are connected by a dashed line to the C-terminal amino acid sequences of the right tree.
Figure 2.
Defining the CRN8 cell death induction domain.
In planta expression of various N-terminal or C-terminal deletions of CRN8, demonstrating the minimal domain necessary for cell death induction. The D2 domain is indicated in yellow, whereas the red portion indicates the position of the functional NLS.
Figure 3.
CRN8 is an active kinase in planta.
(A) An overview of the CRN8 construct, including the catalytic domain (red) and the amino acid positions of Arginine (R) at 469 and Aspartate (D) at 470. (B) The upper panel indicates the protein input for the kinase assay. The lower panel shows an auto-radiogram detecting phosphorylation state of CRN8, CRN8D470N, and CRN8R469A;D470A, with only CRN8 producing a signal. (C) Phosphorylated CRN8 protein was detected in crude plant protein extract (top panel) and in FLAG immuno-purified protein (lower panel) by Pro-Q Diamond phosphoprotein in gel stain. The middle panel shows that all three fusion proteins were present, as detected by FLAG antibody on a Western blot. (D) Macroscopic cell death associated with A. tumefaciens-mediated transient expression of FLAG:CRN8 and kinase-inactive mutants FLAG:CRN8D470N and FLAG:CRN8R469A;D470A in N. benthamiana. Picture was taken five days post A. tumefaciens infiltration in N. benthamiana.
Figure 4.
Identification and role of phosphorylated serines in CRN8 cell death induction.
(A) A cartoon of the CRN8 protein (yellow) with the five phosphorylated serines identified by LC MS/MS denoted by green bars. Portion of CRN8 shown in light yellow was not represented in peptide coverage. Asterisks indicate serines that were included in the CRN8S3xA triple mutant. FLAG sequence (blue portion) and the linker sequence (orange portion) are included. (B) The top panel shows macroscopic cell death associated with A. tumefaciens-mediated transient expression of FLAG:CRN8, FLAG:CRN8D470N, FLAG:CRN8R469A;D470A, FLAG:CRN8S3xA, and FLAG:CRN8S5xA in N. benthamiana. Pictures were taken 8 days after inoculation. The middle panel shows that phosphorylated protein was detected for FLAG:CRN8, FLAG:CRN8S3xA, and FLAG:CRN8S5xA the in FLAG immuno-purified protein by Pro-Q Diamond phosphoprotein in gel stain. The bottom panel shows that all five FLAG immuno-purified fusion proteins were present, as detected by FLAG antibody on a Western blot.
Figure 5.
The kinase dead mutant CRN8R469A;D470A suppresses CRN8-induced cell death.
N. benthamiana leaf displaying CRN8-induced cell death shown five days post infiltration with A. tumefaciens. Different inoculation combinations are shown above for each half of the leaf: CRN8 with GFP (left) and CRN8 with CRN8R469A;D470A (right). To the left of the picture, the five different ratios of each tested combination are indicated.
Figure 6.
The kinase inactive mutant, CRN8R469A;D470A, destabilizes the CRN8 protein.
(A) Overview of the different combinations of Agrobacterium-mediated co-expressed proteins in N. benthamiana leaves. (B) Macroscopic cell death 5 days post infiltration in N. benthamiana leaves; strongest cell death classified as ++ and suppressed cell death as +/−. (C) Western blot of FLAG:CRN8 protein inputs probed with FLAG antibody. (D) Coomassie stain indicating equal loading of protein on Western blot depicted in figure 6C. (E) Western blot of GFP, GFP:CRN8, and GFP:CRN8R469A;D470A signals from crude protein extracts probed with GFP antibody. (F) Coomassie stain indicating equal loading of protein on Western blot depicted in figure 6E.
Figure 7.
(A) Overview of the different combinations of FLAG:CRN8, GFP:CRN8, FLAG:CRN8R469A;D470A, and GFP (indicated by +) proteins, co-expressed in N. benthamiana leaves. (B) Western blot of GFP:CRN8 and GFP protein from GFP immuno-purified protein samples probed with GFP antibody. (C) Western blot of FLAG:CRN8 and FLAG:CRN8R469A;D470A inputs for GFP co-immunopurification experiment probed with FLAG antibody. (D) Co-immunoprecipitation of FLAG:CRN8 and FLAG:CRN8 R469A;D470A in GFP immuno-purified protein samples on a Western blot probed with FLAG antibody. (E) Coomassie stain indicating equal loading of protein on Western blot in figure 7D.
Figure 8.
CRN8 is important for full virulence and CRN8R469A;D470A reduces P. infestans virulence.
(A) The graph shows the growth rate of P. infestans lesions when co-inoculated with GFP:CRN8 or GFP. The Y-axis shows the millimeter change in average lesion diameter (n = 21) between days 4 and 5, while the X-axis shows which construct was infiltrated 2 days post P. infestans inoculation. Errors bars indicate the standard error (p<0.1). (B) The graph shows the growth rate of P. infestans lesions when co-inoculated with GFP:CRN8R469A;D470A or GFP. The Y-axis shows the millimeter change in average lesion diameter (n = 22) between days 5 and 10 post infection, while the X-axis shows which construct was infiltrated before P. infestans inoculation. Errors bars indicate the standard error (p<0.0005).