Forward Genetic In Planta Screen for Identification of Plant-Protective Traits of Sphingomonas sp. Strain Fr1 against Pseudomonas syringae DC3000

Abstract

Sphingomonas sp. strain Fr1 has recently been shown to protect Arabidopsis thaliana against the bacterial leaf pathogen Pseudomonas syringae DC3000. Here, we describe a forward genetic in planta screen to identify genes in Sphingomonas sp. Fr1 necessary for this effect. About 5,000 Sphingomonas sp. Fr1 mini-Tn5 mutants were assayed for a defect in plant protection against a luxCDABE-tagged P. syringae DC3000 derivative in a space-saving 24-well plate system. The bioluminescence of the pathogen was used as the indicator of pathogen proliferation and allowed for the identification of Sphingomonas sp. Fr1 mutants that had lost the ability to restrict pathogen growth before disease symptoms were visible. Potential candidates were validated using the same miniaturized experimental system. Of these mutants, 10 were confirmed as plant protection defective yet colonization competent. The mutants were subsequently evaluated in a previously described standard microbox system, and plants showed enhanced disease phenotypes after pathogen infection relative to those inoculated with the parental strain as a control. However, the disease severities were lower than those observed for control plants that were grown axenically prior to pathogen challenge, which suggests that several traits may contribute to plant protection. Transposon insertion sites of validated mutants with defects in plant protection were determined and mapped to 7 distinct genomic regions. In conclusion, the established screening protocol allowed us to identify mutations that affect plant protection, and it opens the possibility to uncover traits important for in planta microbe-microbe interactions.

Systems-level Proteomics of Two Ubiquitous Leaf Commensals Reveals Complementary Adaptive Traits for Phyllosphere Colonization

“…tomato DC3000. The detailed molecular mechanism of this interaction remains elusive, but several traits might contribute incrementally to plant host protection, including stimulation of plant host immunity and competition for nutrients, especially during the first phase of pathogen invasion into the host-associated microbiota (20,21). The in planta proteome of S. melonis Fr1 indicates utilization of substrates feeding into the TCA cycle including acetate, fatty acid compounds, dicarboxylates and the amino acid alanine.…”

Metabolic footprint of epiphytic bacteria on Arabidopsis thaliana leaves

“…Camalexin biosynthesis genes have been shown to be elicited in leaves by the commensal rhizobacterium Pseudomonas fluorescens SS101 (van de Mortel et al, 2012), with no significant expression in roots. Commensal-induced camalexin production at the site of bacterial colonization has not been previously demonstrated, but is interesting in view of the plant protection effect of S. melonis Fr1 against phytopathogens (Innerebner et al, 2011;Vogel et al, 2012), for which proof for the mode of action at the molecular level is currently lacking. It must be stressed that more data are needed to elucidate the mechanism of plant protection; however, S. melonis-elicited camalexin production might be one element involved in the reported plant protection from P. syringae pv.…”

Versatile Vectors for Efficient Mutagenesis of Bradyrhizobium diazoefficiens and Other Alphaproteobacteria

“…We transformed plasmids pRp_TIE-1-0002 and pRp_TIE-1-0003 into E. coli S17-1 pir and plasmids pSm_Fr1-0002 and pSm_Fr1-0003 into E. coli SM10. We performed matings as described previously (24,25) and counterselected E. coli cells with streptomycin (S. melonis) or high gentamicin concentrations (800 g/ml; R. palustris). We screened exconjugants for double-crossover candidates as described above for B. diazoefficiens and verified their genotypes by PCR.…”