Abstract
Dickeya dadantii is a bacterial plant pathogen that causes soft rot disease on a wide range of host plants. The type III secretion system (T3SS) is an important virulence factor in D. dadantii. Expression of the T3SS is induced in the plant apoplast or in hrp-inducing minimal medium (hrp-MM), and is repressed in nutrient-rich media. Despite the understanding of induction conditions, how individual cells in a clonal bacterial population respond to these conditions and modulate T3SS expression is not well understood. In our previous study, we reported that in a clonal population, only a small proportion of bacteria highly expressed T3SS genes while the majority of the population did not express T3SS genes under hrp-MM condition. In this study, we developed a method that enabled in situ observation and quantification of gene expression in single bacterial cells in planta. Using this technique, we observed that the expression of the T3SS genes hrpA and hrpN is restricted to a small proportion of D. dadantii cells during the infection of potato. We also report that the expression of T3SS genes is higher at early stages of infection compared to later stages. This expression modulation is achieved through adjusting the ratio of T3SS ON and T3SS OFF cells and the expression intensity of T3SS ON cells. Our findings not only shed light into how bacteria use a bi-stable gene expression manner to modulate an important virulence factor, but also provide a useful tool to study gene expression in individual bacterial cells in planta.
Highlights
Dickeya dadantii is a Gram-negative plant pathogenic bacterium that causes soft-rot, stem wilt, and blight diseases on a wide range of economically important crops including potato, carrots, and cabbage
To further understand what occurred at the single-cell level that resulted in the change in overall expression of T3SS in the total bacterial population as determined by quantitative reverse transcription PCR (qRT-PCR), we used the dual fluorescence reporter nptII-gfp-hrpN-mCherry to monitor the transcription of hrpN during disease progression in single cells of D. dadantii
We demonstrated the feasibility of using a dual fluorescence reporter plasmid in combination with single cell technologies to observe and quantify gene expression at the single cell level during the host–microbe interaction, and proved that D. dadantii truly displayed heterogeneity in expressing T3SS genes during infection of potato
Summary
Dickeya dadantii is a Gram-negative plant pathogenic bacterium that causes soft-rot, stem wilt, and blight diseases on a wide range of economically important crops including potato, carrots, and cabbage. Using single cell technology, our previous study demonstrated that the expression of T3SS genes in a bacterial population is heterogeneous under in vitro conditions: multiple T3SS genes including hrpA (encoding the hrp pilus), hrpN (encoding a harpin protein), and dspE (encoding a T3 effector) are highly expressed in only a small proportion of the cells, and are not expressed in the rest of the D. dadantii population under the hrp-inducing conditions (hrp-MM; Zeng et al, 2012) This phenotype of two sub-populations displaying different levels of gene expression in a homogeneous bacterial population was defined as bi-stability (Zeng et al, 2012). The dual reporter we used enables the visualization of the total bacterial population using green fluorescence while monitoring the expression of T3SS genes in single cells with red fluorescence Using this method, we demonstrated the expression dynamics of T3SS genes in single cells of D. dadantii during the early and late stages of infection in various plant tissue types. Our results suggest that by modulating the percentage of the T3SSON cells in the total population and the expression intensity of the T3SSON cells, D. dadantii maintains a higher overall expression of T3SS at the early stages of infection than at the late stage of infection in potato
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