Analysis of the Plant bos1 Mutant Highlights Necrosis as an Efficient Defence Mechanism during D. dadantii/Arabidospis thaliana Interaction

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

Disruption of the Bcchs3a chitin synthase gene in Botrytis cinerea is responsible for altered adhesion and overstimulation of host plant immunity.

International audienceThe fungal cell wall is a dynamic structure that protects the cell from different environmental stresses suggesting that wall synthesizing enzymes are of great importance for fungal virulence. Previously, we reported the isolation and characterization of a mutant in class III chitin synthase, Bcchs3a, in the phytopathogenic fungus Botrytis cinerea. We demonstrated that virulence of this mutant is severely impaired. Here, we describe the virulence phenotype of the cell-wall mutant Bcchs3a on the model plant Arabidopsis thaliana and analyze its virulence properties, using a variety of A. thaliana mutants. We found that mutant Bcchs3a is virulent on pad2 and pad3 mutant leaves defective in camalexin. Mutant Bcchs3a was not more susceptible towards camalexin than the wild-type strain but induced phytoalexin accumulation at the infection site on Col-0 plants. Moreover, this increase in camalexin was correlated with overexpression of the PAD3 gene observed as early as 18 h postinoculation. The infection process of the mutant mycelium was always delayed by 48 h, even on pad3 plants, probably because of lack of mycelium adhesion. No loss in virulence was found when Bcchs3a conidia were used as the inoculum source. Collectively, these data led us to assign a critical role to the BcCHS3a chitin synthase isoform, both in fungal virulence and plant defense response

Disease development in the <i>Arabidopis bos1</i> mutant.

<p>Leaves were inoculated by needle wounding and depositing a 5 µl drop of a 10⁴ cfu/mL <i>D. dadantii</i> (strain 3937) suspension on Col-0 wild type and <i>bos1</i> mutant plants. <b>A:</b> typical maceration symptoms on Col-0 leaves. <b>B:</b> necrotic margin appearing around the maceration zone in Col-0 infected leaves. <b>C:</b> dried maceration zone surrounded by necrotic tissues in an almost totally chlorotic leaf of the <i>bos1</i> mutant. <b>D:</b> systemic necrosis on whole <i>bos1</i> plants 7 dpi. <b>E:</b> whole <i>bos1</i> plants inoculated with mycelium plugs of <i>B. cinerea</i> (strain BD90) 7 dpi. <b>m</b>: macerated tissue; <b>c</b>: chlorotic tissue; <b>g</b>: green tissue; n: necrosis; <b>Inf L</b>: infected leaf. <b>F:</b> kinetics of soft rot progression (top) and necrosis development (bottom) in Col-0 wild type plants (left) and <i>bos1</i> mutant (right). Inoculation of at least 40 Col-0 and <i>bos1</i> 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 <i>bos1</i> (Fisher test comparing the highest score at each day, p<0,05). <b>G: </b><i>In planta</i> growth kinetics of <i>D. dadantii</i> on Col-0 wild type (dash line, triangles) and <i>bos1</i> 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 <i>bos1</i> (Student's t-tests, p<0,01). The experiment has been performed three times with similar results.</p

Enhancement of <i>D. dadantii</i> -induced cell death in <i>bos1</i> leaves.

<p><b>A:</b> 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 <i>bos1</i> 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>B:</b> intracellular oxidative stress 2 and 3 dpi analyzed by DCFH-DA staining of Col-0 wild type and <i>bos1</i> mutant leaves inoculated with <i>D. dadantii</i> after wounding. All experiments have been performed at least three times.</p

Early oxidative stress and protein cross-linking in Col-0 and <i>bos1</i> mutant leaves during <i>D. dadantii</i> infection.

<p>The leaves were inoculated by depositing about 50 bacteria or 5 µl phosphate buffer after needle wounding and staining was performed 1 dpi. <b>A:</b> oxidative stress analyzed by DAB staining of Col-0 wild type and <i>bos1</i> mutant infected leaves. <b>B:</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.</p

<i>D. dadantii</i> induces <i>BOS1</i> gene expression through the secretion of specific proteins.

<p>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 <i>BOS1</i> gene expression was analyzed by quantitative real-time RT-PCR using the <i>BETA-6 TUBULIN</i> as constitutive standard gene. <b>A:</b> analysis of the involvement of bacterial protein secretion systems in the induction of <i>BOS1</i> expression. Tested bacterial strains are 3937 wild type strain, <i>prtE</i> type I secretion system mutant, <i>outC</i> type II secretion system mutant and <i>hrcC</i> type III secretion system mutant. Relative <i>BOS1</i> 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>B:</b> analysis of the involvement of the major pectinases secreted through the type II secretion system in the induction of <i>BOS1</i> expression. Tested bacterial strains are 3937 wild type strain, <i>Δpel</i> mutant strain deficient for the production of the five major pectinases PelA to PelE, <i>pelA</i> mutant strain deficient for the production of the PelA pectinase, <i>pelBC</i> mutant strain deficient for the production of the PelB and PelC pectinases, <i>pelDE</i>, mutant strain deficient for the production of the PelD and PelE pectinases. Relative <i>BOS1</i> 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.</p

Bacterial strains, fungus and primers used in this study.

<p>Bacterial strains, fungus and primers used in this study.</p