Fig 1.
Symptoms and bacterial growth in plants inoculated with Clavibacter michiganensis (Cm) isolates.
Four-leaf stage “Moneymaker” tomato plants (A, C) or three-leaf stage “Black Queen” eggplants (B, D) were inoculated with the indicated Cm isolates or water control (mock) by puncturing the stem area between the cotyledons with a wooden toothpick incubated in Cm solution (5 × 107 CFU/ml). (A, B) Graphs represent the distribution of symptom severity in response to each isolate in at least eight plants taken from at least two experimental repeats. Symptoms severity were scored at 14 days post inoculations (dpi) according to the percentage of leaves displaying wilt (tomato) or leaf blotch (eggplant) symptoms by the following scale: 0 = no wilting/leaf blotch, 1 = 1–25%, 2 = 25–50%, 3 = 50–100%. (C, D) Stem bacterial populations 1 cm above the inoculation sites were quantified at 14 dpi. Lower and upper quartiles are marked at the margins of the boxes. Central lines, "×" and “o” represent medians, means and data points of at least eight biological repeats collected from at least two independent experiments. Boxes marked in red represent isolates that caused symptoms in tomato but not in eggplant, boxes marked in purple represent isolates that caused symptoms in tomato and in eggplant, and boxes marked in blue represent isolate that failed to cause symptoms in either tomato or eggplant. All depicted data were analyzed using one-way ANOVA followed by post-hoc Tukey HSD test. Letters indicate similarity in disease severity and bacterial populations (Tukey HSD test, p value ≤ 0.05).
Table 1.
Virulence analyses of Cm isolates.
Fig 2.
Elicitation of the hypersensitive response (HR) in eggplant leaves by Clavibacter michiganensis (Cm) isolates.
Five to six leaf stage “Black Queen” eggplant leaves were infiltrated (108 CFU/ml) with the indicated Cm isolates. Pictures were taken 48 h post infiltration. Infiltrated area are marked in colored dotted lines. Red lines represent isolates that caused symptoms in tomato but not in eggplant, purple lines represent isolates that caused symptoms in tomato and in eggplant, and blue lines represent isolate that failed to cause symptoms in either tomato or eggplant. Pictures are representatives of one out of at least 10 repeats from at least two independent experiments.
Fig 3.
Tomato non-pathogenic Clavibacter michiganensis (Cm) isolates lack the chp/tomA pathogenicity island.
(A) Phylogenetic tree of Cm isolates used in this study. The tree was produced using the M1CR0B1AL1Z3R web server (https://microbializer.tau.ac.il/) and is based on maximum-likelihood multiple sequence alignments of 129 core genes under default features and visualized by PhyD3. Genomes used for the analysis are 40 genomes of Cm isolates characterized in this study; references Cm strains UF1, LMG7333, NCPPB382, CMM84, Cmm_21, VQ143, VQ28, VL527, A5747, N.P., MSF322, and CMM04; and references strains of C. sepedonicus ATCC331133, C. capsici PF008, C. nebraskensis NCPPB2581, C. insidiosus ATCC10253, C. tessellarius ATCC33566, C. zhangzhiyongii DM1, C. phaseoli LPPA982, and C. californiensis CFBP8216. Curtobacterium flaccumfaciens Cff1037 was used as an outgroup. Isolates which were pathogenic on tomato but not pathogenic on eggplant are marked with a red dot, isolates which were pathogenic on tomato and eggplant are marked with a purple dot, and isolates which were non-pathogenic on tomato and eggplant are marked with a blue dot. (B) Whole genome alignment of the tomato non-pathogenic Cm isolates C3, C4, C30, C31, and C61 was performed against CDS of Cm strain NCPPB382 chromosome (NCBI GenBank: AM711867), pCM1 plasmid (AM711865) and pCM2 plasmid (AM711866) and visualized with BLAST atlas analysis in Gview server (https://server.gview.ca/) using default features. The chp/tomA genomic island (positions 42,081–162,441 in the NCPPB382 chromosome) is labeled. (C) Physical maps of the area surrounding the chp/tomA island in the tomato-pathogenic reference strains NCPPB382 (AM711867), UF1 (NZ_CP033724), and VL527 (NZ_CP047054) and representative corresponding regions in the tomato non-pathogenic isolates C61 [JASBOJ000000000, contig 16. Similar gene synteny was observed in C3 (JASBQC000000000, contig 13, 59,987–70,253) and C4 (JASBQB000000000, contig 14, 59,985–70,252)], and C30 [JASBPU000000000, contig 9. Similar gene synteny was observed in C31 (JASBPN000000000, contig 9, 36,376–25,484)]. Predicted ORFs are marked in arrows supplemented with locus tags in the corresponding genomes. Similar colors indicates the ORFs share DNA sequence identify of >97%, "/" indicates an ORF was disrupted by a frameshift mutation. The chp/tomA and direct repeat region are marked. The first ORF and the two last ORFs of the chp/tomA region are labeled in shades of yellow. The two ORFs encoded within the direct repeat region are labeled in shades of purple.
Fig 4.
The ChpG allelic variants are differentially recognized in eggplant.
(A) Schematic representation of the four ChpG protein variants depicted in S6 Fig. Signal peptide region, predicted by SignalP-5.0 (https://services.healthtech.dtu.dk/services/SignalP-5.0/) is labeled in yellow. Alpha-lytic serine protease domain, predicted by NCBI conserved domain search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) is labeled in green. Amino acid polymorphic sites are marked in black lines. (B) Model represents rotated forms of the predicted 3D structure of ChpG variant A (ChpGA). Structure prediction was conducted by Alphafold (https://alphafold.ebi.ac.uk/entry/A5CLZ4, UniPort num’ A5CLZ4) and visualized by Mol* (https://molstar.org/viewer/). Amino acid polymorphic sites unique to ChpGA, ChpGC and ChpGD are respectively marked in light green, magenta and blue. The H93, D119, and S231 amino acid residues representing the serine protease catalytic triad are respectively marked in brown, red and yellow. (C, D) Black Queen eggplant leaves were infiltrated with 10 mM MgCl2 (mock) or suspensions (108 CFU/ml) of Cm101, and Cm101ΩchpG clones expressing the indicated chpG variants or empty vector control (EV). (C) Representative picture was taken 48 h post infiltration (hpi). (D) Cell death was quantified by ion leakage at 36 hpi. Lower and upper quartiles are marked at the margins of the boxes. Central lines and “o” represent medians and data points of 21 biological repeats collected from three independent experiments. "*" indicates significant differences (Mann–Whitney U test, p-value ≤ 0.05) from Cm101ΩchpG + EV.
Fig 5.
The ChpGC variant does not elicit HR in eggplant.
(A) Mature ChpG variants, lacking the predicted secretion sequence, were fused to a maltose-binding protein (MBP) tag, purified from E. coli, and visualized by SDS-PAGE using Coomassie blue staining (upper panel) and western blot analysis using anti MBP antibody (lower panel). (B, C) Purified proteins (0.01 μg/ml) or MgCl2 control (mock) were infiltrated into “Black Queen” eggplant leaves. (B) Representative leaf was photographed 36 hours post infiltration (hpi). (C) Cell death was quantified by ion leakage at 24 and 36 hpi. Lower and upper quartiles are marked at the margins of the boxes. Central lines and “o” represent medians and data points of at least 12 biological repeats collected from three independent experiments. "*" indicates a significant difference (Mann–Whitney U test, p-value ≤ 0.05) from mock control.
Fig 6.
The ChpGC variant does not restrict Clavibacter michiganensis (Cm) from colonizing eggplant.
(A, B) Five to six leaf stage “Black Queen” eggplant leaves were infiltrated (108 CFU/ml) with Cm101, Cm101ΩchpG and CmC48 expressing the indicated ChpG variants or empty vector control (EV). (A) Picture was taken at 36 hours post inoculations (hpi). (B) Cell death was quantified by ion leakage at 24 and 36 hpi. Lower and upper quartiles are marked at the margins of the boxes. Central lines and “o” represent medians and data points of at least 12 (24 h) or 17 (36 h) biological repeats collected from at least two (24 h) or three (36 h) independent experiments. "*" indicates a significant difference (Mann–Whitney U test, p-value ≤ 0.05) from Cm101ΩchpG (left panel) or CmC48 (right panel). (C, D, E, F) Three-leaf stage “Black Queen” eggplants were inoculated with the indicated Cm strains or water control (mock) by puncturing the stem area between the cotyledons with a wooden toothpick incubated in Cm solution (5 × 107 CFU/ml). (C, D) Representative plants were photographed 14 days post inoculation (dpi). (E) Leaf blotch symptoms were quantified at 14 dpi according to the following scale: 0 = no leaf blotch, 1 = 1–25%, 2 = 25–50%, 3 = 50–100%. Graph depicts the symptom distribution in at least 15 plants pooled from at least three independent experiments. “*" indicates the score distribution is different from Cm101ΩchpG (left panel) or CmC48 (right panel) (Pearson’s chi-squared test, p-value ≤ 0.05). (F) Stem bacterial populations 1 cm above the inoculation sites were quantified at 14 dpi. Lower and upper quartiles are marked at the margins of the boxes. Central lines and “o” represent medians and data points of 15 biological repeats collected from at least three independent experiments. "*" indicates a significant difference (Mann–Whitney U test, p-value ≤ 0.05) from Cm101ΩchpG (left panel) or CmC48 (right panel).
Fig 7.
Differential recognition of ChpG variants is linked to a single polymorphic amino acid.
(A, B) Five to six leaf stage “Black Queen” eggplant leaves were infiltrated (108 CFU/ml) with Cm101, Cm101ΩchpG expressing the indicated ChpG variants or empty vector control (EV). (A) Picture was taken at 36 hours post inoculation (hpi). (B) Cell death was quantified by ion leakage at 24 and 36 hpi. Lower and upper quartiles are marked at the margins of the boxes. Central lines and “o” represent medians and data points of at least 9 (24 h) or 11 (36 h) biological repeats collected from two independent experiments. "*" indicates significant difference (Mann–Whitney U test, p-value ≤ 0.05) from Cm101ΩchpG. (C, D, E) Three-leaf stage “Black Queen” eggplants were inoculated with the indicated Cm strains or water control (mock) by puncturing the stem area between the cotyledons with a wooden toothpick incubated in Cm solution (5 × 107 CFU/ml). (C) Representative plants were photographed 14 days post inoculations (dpi). (D) Leaf blotch symptoms were quantified at 14 dpi according to the following scale: 0 = no leaf blotch, 1 = 1–25%, 2 = 25–50%, 3 = 50–100%. Graph depicts the symptom distribution in at least 15 plants pooled from three independent experiments. “*" indicates the score distribution is different from Cm101ΩchpG (Pearson’s chi-squared test, p-value ≤ 0.05). (E) Stem bacterial populations 1 cm above the inoculation sites were quantified at 14 dpi. Lower and upper quartiles are marked at the margins of the boxes. Central lines and “o” represent medians and data points of 16 biological repeats collected from three independent experiments. "*" indicates a significant difference (Mann–Whitney U test, p-value ≤ 0.05) from Cm101ΩchpG.
Fig 8.
Immune recognition of ChpG variants in Mirabilis jalapa.
(A, B, C) Eight to ten leaf stage Mirabilis jalapa leaves were infiltrated (108 CFU/ml) with Cm101ΩchpG expressing the indicated ChpG variants or empty vector control (EV). (A) Representative leaf was photographed at 96 hours post inoculations (hpi). (B) HR was monitored at 48 and 96 hpi in the infiltrated areas and each infiltration point received a HR classification (HR or no HR). HR frequencies of 15 biological repeats collected from three independent experiments are presented in a stacked bar graph. “*" indicates the score distribution is different from Cm101ΩchpG + EV (Pearson’s chi-squared test, p-value ≤ 0.05). (C) Cell death was quantified by ion leakage at 96 hpi. Lower and upper quartiles are marked at the margins of the boxes. Central lines and “o” represent medians and data points of at least 12 biological repeats collected from two independent experiments. "*" indicates a significant difference (Mann–Whitney U test, p-value ≤ 0.05) from Cm101ΩchpG + EV. (D) Mirabilis jalapa leaves were infiltrated with the indicted MBP fused ChpG variants (0.01 μg/ml) or MgCl2 (mock). Representative leaf was photographed 3 days later. The image represents 18 biological replicates across three independent experiments, all showcasing comparable results.
Fig 9.
Serine hydrolase activity assays in ChpG variants failed to detect enzymatic activity.
(A) Mature ChpG variants, lacking the predicted secretion sequence, were fused to a maltose-binding protein (MBP) tag, purified from E. coli, and visualized by SDS-PAGE using Coomassie blue staining. (B) N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide cleavage assays were conducted using 1 μg of the indicated MBP fusion proteins or 0.1 μg proteinase K. Data represents the averages and standard divisions of three technical repeats. (C) Azocasein hydrolysis assays were conducted using 1 μg of the indicated MBP fusion proteins or 0.1 μg proteinase K. Data represents the averages and standard divisions of at least four technical repeats. (D, E, F) Serine hydrolase activity using the TAMRA-FP serine hydrolase probe was assessed with 0.1 (D) or 1 (E, F) μg proteinase K (positive control), 1 μg of the indicated MBP fusion proteins (D), total protein extracts, and supernatants of Cm101ΔPAI cultures introduced with pHN216-based plasmids expressing the indicated chpG variants under the pCMP1 promoter fused to an HA tag or an empty vector control (EV) (E), or total protein extracts from Nicotiana benthamiana leaves infiltrated with Agrobacterium carrying vectors aimed at transient expression of the indicated mature chpG variants fused to the 33 aa secretion signal of NtPR1 at the N-terminus and to an HA tag at the C-terminus under the control of the CaMV35S promoter or the 33 aa secretion signal of NtPR1 alone (EV) (F). Left panels (D, E, F): Total protein was visualized by Coomassie blue staining. Middle panels (D, E, F): Active site fluorescent binding was visualized by fluorescent imaging (530 nm excitation, 554 nm emission). Right panels (E, F): Confirmation of expression of the ChpG variants by western blot using anti-HA antibody. Experiments were repeated Twice (B, C) or three times (D, E, F) with similar results.