A straightforward and reliable method for bacterial in planta transcriptomics: application to the Dickeya dadantii/Arabidopsis thaliana pathosystem.

Transcriptome analysis of bacterial pathogens is a powerful approach to identify and study the expression patterns of genes during host infection. However, analysis of the early stages of bacterial virulence at the genome scale is lacking with respect to understanding of plant-pathogen interactions and diseases, especially during foliar infection. This is mainly due to both the low ratio of bacterial cells to plant material at the beginning of infection, and the high contamination by chloroplastic material. Here we describe a reliable and straightforward method for bacterial cell purification from infected leaf tissues, effective even if only a small amount of bacteria is present relative to plant material. The efficiency of this method for transcriptomic analysis was validated by analysing the expression profiles of the phytopathogenic enterobacterium Dickeya dadantii, a soft rot disease-causing agent, during the first hours of infection of the model host plant Arabidopsis thaliana. Transcriptome profiles of epiphytic bacteria and bacteria colonizing host tissues were compared, allowing identification of approximately 100 differentially expressed genes. Requiring no specific equipment, cost-friendly and easily transferable to other pathosystems, this method should be of great interest for many other plant-bacteria interaction studies.

PecS is an important player in the regulatory network governing the coordinated expression of virulence genes during the interaction between Dickeya dadantii 3937 and plants.

Successful infection of a pathogen relies on the coordinated expression of numerous virulence factor-encoding genes. In plant-bacteria interactions, this control is very often achieved through the integration of several regulatory circuits controlling cell-cell communication or sensing environmental conditions. Dickeya dadantii (formerly Erwinia chrysanthemi), the causal agent of soft rot on many crops and ornamentals, provokes maceration of infected plants mainly by producing and secreting a battery of plant cell wall-degrading enzymes. However, several other virulence factors have also been characterized. During Arabidopsis infection, most D. dadantii virulence gene transcripts accumulated in a coordinated manner during infection. This activation requires a functional GacA-GacS two-component regulatory system but the Gac system is not involved in the growth phase dependence of virulence gene expression. Here we show that, contrary to Pectobacterium, the AHL-mediated ExpIR quorum-sensing system does not play a major role in the growth phase-dependent control of D. dadantii virulence genes. On the other hand, the global regulator PecS participates in this coordinated expression since, in a pecS mutant, an early activation of virulence genes is observed both in vitro and in planta. This correlated with the known hypervirulence phenotype of the pecS mutant. Analysis of the relationship between the regulatory circuits governed by the PecS and GacA global regulators indicates that these two regulators act independently. PecS prevents a premature expression of virulence genes in the first stages of colonization whereas GacA, presumably in conjunction with other regulators, is required for the activation of virulence genes at the onset of symptom occurrence.

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

The 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.

Botrytis cinerea virulence is drastically reduced after disruption of chitin synthase class III gene (Bcchs3a).

Botrytis cinerea is an important phytopathogenic fungus requiring new methods of control. Chitin biosynthesis, which involves seven classes of chitin synthases, could be an attractive target. A fragment encoding one of the class III enzymes was used to disrupt the corresponding Bcchs3a gene in the B. cinerea genome. The resulting mutant exhibited a 39% reduction in its chitin content and an 89% reduction in its in vitro chitin synthase activity, compared with the wild-type strain. Bcchs3a mutant was not affected in its growth in liquid medium, neither in its production of sclerotia, micro- and macroconidia. In contrast, the mutant Bcchs3a was severely impaired in its growth on solid medium. Counterbalancing this defect in radial growth, Bcchs3a mutant presented a large increase in hyphal ramification, resulting in an enhanced aerial growth. Observations by different techniques of microscopy revealed a thick extracellular matrix around the hyphal tips. Moreover, Bcchs3a mutant had a largely reduced virulence on Vitis vinifera and Arabidopsis thaliana leaves.