PMC

(A) Interaction in a yeast-two hybrid assay between proteins in the Bti9 clade with AvrPtoB or its variants. +, a positive interaction (blue yeast patches observed in the assay); −, weak or no interaction (white or faint blue yeast patches). (B) Expression of Bti9 and its homologs in yeast was confirmed by protein blotting using an anti-LexA antibody.

(A) Interaction in a yeast two-hybrid assay between Bti9 or Pto and AvrPtoB or its variants. A non-specific AvrPtoB tomato-interacting protein, Bti6, encoding a putative lactate dehydrogenase was used as a positive control (). Blue patches indicate positive interactions. Photographs were taken 24 hours after yeast transformants were streaked on –Ura –His –Trp plates (). (B) Expression of AvrPtoB and its variants as bait proteins in yeast cells that also express Bti9 was confirmed by Western blotting using an anti-LexA antibody.

(A) Close-up of the crystal structure of the complex between AvrPtoB (in turquoise) and Pto (in red) (). The interface with Pto that is specific to AvrPtoB is shown in the left panel and the interface shared by both AvrPtoB and AvrPto is shown in the right panel. Residues at which amino acid substitutions were made are shown and their positions indicated in different colors (). (B) Interactions in a yeast two-hybrid assay of Bti9 or Pto with AvrPtoB_1-307 and variants. Blue patches indicate a positive interaction. A non-specific AvrPtoB tomato-interacting protein, Bti6, encoding a putative lactate dehydrogenase was used as a positive control (). (C) Immunoblot analysis with anti-HA antibody in the bottom panel shows similar expression in yeast of wild-type AvrPtoB_1-307 and the variant proteins from the prey vector.

The top panel in each part shows a phosphor-image of the kinase assay. The bottom panel in parts B and C shows the Coomassie-stained gel that was used for phosphor-imaging of the kinase assay. (A) Bti9 encodes an active kinase. A maltose-binding protein (MBP) fusion to Bti9 weakly autophosphorylated and strongly trans-phosphorylated myelin basic protein (MyBP). Substitution of Y to D at amino acid 489 abolished Bti9 kinase activity. Reactions without MyBP were used as controls. The Coomassie-stained gel shows equal abundance of Bti9 or Bti9(Y489D) protein (barely visible because only 200 ng was used per lane). (B) AvrPtoB and AvrPtoB_1-307 inhibited Bti9 kinase activity. Addition of GST-AvrPtoB or GST-AvrPtoB_1-307 fusion proteins to the Bti9 kinase assays reduced the ability of Bti9 to phosphorylate MyBP in a dose-dependent manner. Top panel: first lane shows phosphorylation of MyBP by Bti9 without addition of AvrPtoB or AvrPtoB_1-307. The next six lanes show the assay with addition of 2.5, 5.0, or 7.5 μg of GST-AvrPtoB or GST-AvrPtoB_1-307, respectively. The last lane shows addition of GST alone did not inhibit Bti9 kinase activity. (C) The F173A substitution abolished the ability of AvrPtoB to inhibit Bti9 kinase activity. Top panel: first lane shows phosphorylation of MyBP by Bti9 without addition of AvrPtoB. The next six lanes show the assay with addition of 2.5, 5.0, or 7.5 μg of GST-AvrPtoB or GST-AvrPtoB(F173A), respectively. The last lane shows addition of GST alone did not inhibit Bti9 kinase activity.

(A) Disease symptoms of RG-prf3 plants 4 days after inoculation with Pst strains DC3000ΔavrPtoΔavrPtoBΔhopQ1-1ΔfliC carrying either empty vector (pCPP45) or plasmids expressing AvrPtoB, AvrPtoB_1-387, or AvrPtoB_1-387(F173A). Red arrows point to enhanced necrosis observed on lower leaves, a characteristic of AvrPtoB virulence (). (B) Bacterial populations in leaves of plants as shown in (A). Pst strains were infiltrated into RG-prf3 plants using an inoculum of 3 × 10⁴ cfu/mL. Each treatment represents the mean of four plants and bars show the standard error. Experiments were repeated three times with similar results. Means with different letters were significantly different based on a Tukey-Kramer HSD test (α= 0.05). (C) Expression of AvrPtoB, AvrPtoB_1-387, or AvrPtoB_1-387(F173A) in DC3000ΔavrPtoΔavrPtoBΔhopQ1-1ΔfliC carrying corresponding plasmids grown in minimal medium under hrp induction conditions () confirmed using a polyclonal anti-AvrPtoB antibody (). The lower band in the AvrPtoB lane is likely a degradation product.

(A) Transcript abundance of Bti9 and its three closest tomato homologs was reduced in CF5 and CF26 transgenic lines carrying the hpBti9 construct. Expression was analyzed by qRT-PCR using SlEF1α as a normalization control. Similar results were obtained using SlATPase normalization. The relative ratio of expression sets 1.0 as the normalized expression of the gene in wild-type plants. Each bar represents the mean of 8 plants and the bars show the standard error of the mean (σ^E). Means with different letters were significantly different based on a Tukey-Kramer HSD test (α = 0.05). (B) Disease symptoms of plants as shown in (A) where either hpBti9 was lost by segregation (termed ‘azygous’ CF26; aCF26) or where hpBti9 was present (hpBti9-CF26) thus transcript abundance of Bti9 was reduced. The photos were taken 4 days after vacuum infiltration of plants with DC3000ΔavrPtoΔ avrPtoBΔ hopQ1-1ΔfliC (3 × 10⁴ cfu/mL). Red arrows in the lower panel (left) point to necrosis of lower leaves, which is a characteristic symptom of AvrPtoB virulence. Right panel shows close-up of lower leaves from aCF26 or from hpBti9-CF26. (C) Bacterial populations in leaves of aCF26 or hpBti9-CF26 tomato plants after inoculation of DC3000ΔavrPtoΔ avrPtoB ΔhopQ1-1Δ fliC as described in (B). Each treatment represents the mean of 5 plants and the bar shows the standard error. The asterisk denotes the difference of bacterial populations in leaves of aCF26 and hpBti9-CF26 was significant as tested using JMP software package (version 7, http://www.jmp.com/; Cary, NC) by a Student’s T test (α = 0.05). This experiment was performed three times with similar results.