Analysis of the plant bos1 mutant highlights necrosis as an efficient defence mechanism during D. dadantii/Arabidospis thaliana interaction

Abstract

Dickeya dadantii is a broad host range phytopathogenic bacterium provoking soft rot disease on many plants including Arabidopsis. We showed that, after D. dadantii infection, the expression of the Arabidopsis BOS1 gene was specifically induced by the production of the bacterial PelB/C pectinases able to degrade pectin. This prompted us to analyze the interaction between the bos1 mutant and D. dadantii. The phenotype of the infected bos1 mutant is complex. Indeed, maceration symptoms occurred more rapidly in the bos1 mutant than in the wild type parent but at a later stage of infection, a necrosis developed around the inoculation site that provoked a halt in the progression of the maceration. This necrosis became systemic and spread throughout the whole plant, a phenotype reminiscent of that observed in some lesion mimic mutants. In accordance with the progression of maceration symptoms, bacterial population began to grow more rapidly in the bos1 mutant than in the wild type plant but, when necrosis appeared in the bos1 mutant, a reduction in bacterial population was observed. From the plant side, this complex interaction between D. dadantii and its host includes an early plant defence response that comprises reactive oxygen species (ROS) production accompanied by the reinforcement of the plant cell wall by protein cross-linking. At later timepoints, another plant defence is raised by the death of the plant cells surrounding the inoculation site. This plant cell death appears to constitute an efficient defence mechanism induced by D. dadantii during Arabidopsis infection.

Figure 1. D. dadantii induces BOS1 gene expression through the secretion of specific proteins.

Six-week-old Col-0 wild type plants were inoculated by immersion into phosphate buffer or into 5.10⁷ cfu/mL bacterial suspensions. Rosettes were harvested at the time points indicated at the bottom and BOS1 gene expression was analyzed by quantitative real-time RT-PCR using the BETA-6 TUBULIN as constitutive standard gene. A: analysis of the involvement of bacterial protein secretion systems in the induction of BOS1 expression. Tested bacterial strains are 3937 wild type strain, prtE type I secretion system mutant, outC type II secretion system mutant and hrcC type III secretion system mutant. Relative BOS1 transcript levels were expressed according to the reference condition (0 hour post infection) set to 1 for each genotype. This result is a representative example out of three biological replicates. B: analysis of the involvement of the major pectinases secreted through the type II secretion system in the induction of BOS1 expression. Tested bacterial strains are 3937 wild type strain, Δpel mutant strain deficient for the production of the five major pectinases PelA to PelE, pelA mutant strain deficient for the production of the PelA pectinase, pelBC mutant strain deficient for the production of the PelB and PelC pectinases, pelDE, mutant strain deficient for the production of the PelD and PelE pectinases. Relative BOS1 transcript levels were expressed according to the reference condition (0 hour post infection) set to 1 for each genotype. This result is a representative example out of two biological replicates.

Figure 2. Disease development in the Arabidopis bos1 mutant.

Leaves were inoculated by needle wounding and depositing a 5 µl drop of a 10⁴ cfu/mL D. dadantii (strain 3937) suspension on Col-0 wild type and bos1 mutant plants. A: typical maceration symptoms on Col-0 leaves. B: necrotic margin appearing around the maceration zone in Col-0 infected leaves. C: dried maceration zone surrounded by necrotic tissues in an almost totally chlorotic leaf of the bos1 mutant. D: systemic necrosis on whole bos1 plants 7 dpi. E: whole bos1 plants inoculated with mycelium plugs of B. cinerea (strain BD90) 7 dpi. m: macerated tissue; c: chlorotic tissue; g: green tissue; n: necrosis; Inf L: infected leaf. F: kinetics of soft rot progression (top) and necrosis development (bottom) in Col-0 wild type plants (left) and bos1 mutant (right). Inoculation of at least 40 Col-0 and bos1 plants was performed on a single leaf per plant as previously described. Symptoms were scored during 6 days using 4 step scales as follows. Maceration scale: stage 0, no symptoms; stage 1, maceration around the bacterial suspension droplet; stage 2, spreading maceration; stage 3, maceration of the whole limb. Necrosis scale: stage 0, no necrosis; stage 1, necrosis surrounding the maceration zone (B); stage 2, necrosis of the whole infected leaf (C); stage 3, systemic necrosis (D). Asterisks indicate significant differences between Col-0 and bos1 (Fisher test comparing the highest score at each day, p<0,05). G: In planta growth kinetics of D. dadantii on Col-0 wild type (dash line, triangles) and bos1 mutant (dotted line, circles). Plants were inoculated as previously described. Each point corresponds to the average of at least 20 numerations and bars correspond to the standard errors. Asterisks indicate significant differences between Col-0 and bos1 (Student's t-tests, p<0,01). The experiment has been performed three times with similar results.

Figure 3. Early oxidative stress and protein cross-linking in Col-0 and bos1 mutant leaves during D. dadantii infection.

The leaves were inoculated by depositing about 50 bacteria or 5 µl phosphate buffer after needle wounding and staining was performed 1 dpi. A: oxidative stress analyzed by DAB staining of Col-0 wild type and bos1 mutant infected leaves. B: analysis of protein cross-linking. Coomassie staining of leaf cells around the inoculated wound without SDS pre-treatment (center) or with SDS-removal of unbound proteins (right). Leaf cell structures and the presence of bacteria (indicated by arrows) were observed using toluidine blue stained sections (left). Plant genotype and inoculum are indicated on the left of the pictures. Bars represent 50 µm. All experiments have been performed at least three times.

Figure 4. Enhancement of D. dadantii -induced cell death in bos1 leaves.

A: trypan blue staining of dead cells after 1–3 dpi. Maceration and necrosis symptoms were photographed (top) and the leaves were stained with trypan blue (bottom). One representative leaf of the eight stained in each case is presented. Col-0 and bos1 leaves were inoculated, after needle wounding, by depositing 5 µl of buffer or 5 µl of a 10⁴ cfu/mL 3937 wild type bacterial strain suspension. B: intracellular oxidative stress 2 and 3 dpi analyzed by DCFH-DA staining of Col-0 wild type and bos1 mutant leaves inoculated with D. dadantii after wounding. All experiments have been performed at least three times.