High‐throughput quantitative luminescence assay of the growth in planta of Pseudomonas syringae chromosomally tagged with Photorhabdus luminescens luxCDABE

Abstract

SummaryBioluminescent strains of the Arabidopsis thaliana pathogens Pseudomonas syringae pathovar (pv.) tomato and pv. maculicola were made by insertion of the luxCDABE operon from Photorhabdus luminescens into the P. syringae chromosome under the control of a constitutive promoter. Stable integration of luxCDABE did not affect bacterial fitness, growth in planta or disease outcome. Luminescence accurately and reliably reported bacterial growth in infected Arabidopsis leaves both with a fixed inoculum followed over time and with varying inocula assayed at a single time point. Furthermore, the bioluminescence assay could detect a small (1.3-fold) difference in bacterial growth between different plant genotypes with a precision comparable to that of the standard plate assay. Luminescence of luxCDABE-tagged P. syringae allows rapid and convenient quantification of bacterial growth without the tissue extraction, serial dilution, plating and manual scoring involved in standard assays of bacterial growth by colony formation in plate culture of samples from infected tissue. The utility of the bioluminescence assay was illustrated by surveying the 500-fold variation in growth of the universally virulent P. syringae pv. maculicola ES4326 among more than 100 Arabidopsis ecotypes and identification of two quantitative trait loci accounting for 48% and 16%, respectively, of the variance of basal resistance to P. syringae pv. tomato DC3000 in the Col-0 · Fl-1 F 2 population. Luminescence assay of bacteria chromosomally tagged with luxCDABE should greatly facilitate the genetic dissection of quantitative differences in gene-for-gene, basal and acquired disease resistance and other aspects of plant interactions with bacterial pathogens requiring high-throughput assays or large-scale quantitative screens.

Next-Generation Systemic Acquired Resistance

“…S2B). Similar results were obtained after inoculation with a bioluminescent strain of PstDC3000 (PstDC3000-lux; Fan et al, 2008): Whereas P 1 wild-type plants developed less bioluminescence and fewer disease symptoms than C 1 wild-type plants, no such differences were observed between P 1 and C 1 progeny from the npr1-1 mutant (Supplemental Fig. S4).…”

“…PstDC3000 assays were performed with a bioluminescent luxCDABEtagged strain (PstDC3000-lux; Fan et al, 2008). Five-week-old plants were inoculated by dipping the leaves in a bacterial suspension containing 10 8 colony-forming units/mL in 10 mM MgSO 4 and 0.01% (v/v) Silwet L-77.…”

Two Different Banana NPR1-Like Coding Sequences Confer Similar Protection Against Pathogens in Arabidopsis

“…This facilitates in planta quantification of bioluminescent Pst-DC3000-Lux bacteria due to the presence of the LuxCDABE operon from Photorhabdus luminescens inserted into the P. syringae chromosome (Fan et al, 2008). During the non-SAR induced treatment (mock/PstDC3000- Fig.…”

“…Growth of Pst-DC3000-Lux was determined quantitatively 48 h after the secondary challenge using 8mm leaf discs excised from Pst-DC3000-Lux-infiltrated leaves as described by Fan et al (2008). A total of 12 plants were used for each time point.…”

Abscisic Acid Has a Key Role in Modulating Diverse Plant-Pathogen Interactions

“…A collection of 8,000 activation tagged T1 plants in the NahG background were screened by hand-infiltration with virulent P. syringae pv maculicola ES4326 (Psm) chromosomally tagged with luxCDABE (Fan et al, 2008). While no mutant was found that enhanced basal resistance in the absence of SA, a mutant with increased disease severity was identified.…”