Consequences of flagellin export through the type III secretion system of Pseudomonas syringae reveal a major difference in the innate immune systems of mammals and the model plant Nicotiana benthamiana
Version of Record online: 21 NOV 2012
© 2012 Blackwell Publishing Ltd
Volume 15, Issue 4, pages 601–618, April 2013
How to Cite
Wei, H.-L., Chakravarthy, S., Worley, J. N. and Collmer, A. (2013), Consequences of flagellin export through the type III secretion system of Pseudomonas syringae reveal a major difference in the innate immune systems of mammals and the model plant Nicotiana benthamiana. Cellular Microbiology, 15: 601–618. doi: 10.1111/cmi.12059
- Issue online: 14 MAR 2013
- Version of Record online: 21 NOV 2012
- Accepted manuscript online: 29 OCT 2012 01:43AM EST
- Manuscript Accepted: 16 OCT 2012
- Manuscript Revised: 28 SEP 2012
- Manuscript Received: 22 JUN 2012
- National Science Foundation Plant Genome Research Program. Grant Number: IOS-1025642
Fig. S1. The shcA-hopA1 deletion in pCPP6225 eliminates the suppressive effect of HopA1 on P. fluorescens Pf0-1 elicitation of ROS in N. benthamiana. pHIR11 carries a portion of the tripartite pathogenicity island of P. syringae pv. syringae 61 comprising a complete hrp/hrc gene cluster flanked by shcA-hopA1 in the exchangeable effector locus and a fragment of the avrE effector gene in the conserved effector locus. Sequential deletions from pHIR11 of shcA-hopA1 and the residual avrE fragment produced plasmids pCPP6225 and pCPP6227 respectively. Pf0-1 carrying these plasmids was tested and analysed for ROS elicitation activity as described in 1. No effect of the avrE fragment was observed.
Fig. S2. P. fluorescens Pf0-1 and Pto DC3000 ΔfliC and ΔflgGHI mutants are deficient in motility on semisolid agar plates. Cells were inoculated with a toothpick from an overnight KB agar plate onto a swim plate (KB plus 0.3% agar) and photographed after a 24 h incubation at 30°C.
A. P. fluorescens Pf0-1 and its derivatives: 1, Pf0-1; 2, Pf0-1 ΔfliC; 3, Pf0-1 ΔflgGHI.
B. Pto DC3000 and its derivatives: 1, Pto DC3000; 2, DC3000 ΔfliC; 3, DC3000 ΔflgGHI; 4, DC3000 ΔfliC ΔflgGHI; 5, DC3000 ΔhrcQ-U; 6, DC3000 ΔhrcQ-U ΔflgGHI; 7, DC3000 ΔhrcQ-U ΔfliC; 8, DC3000 ΔhrpK1 ΔhrpZ1::nptII ΔhrpW1::Spr ΔhopAK1 ΔhopP1 (Trans−); 9, DC3000 Tran− ΔfliC; 10, DC3000 Tran− ΔflgGHI; 11, DC3000 Tran− ΔflgGHI ΔfliC.
Fig. S3. Changes in the relative elicitation of ROS and functional MTI by P. fluorescens Pf01-1 derivatives at two levels of inoculum corroborate the importance of FliC in ROS elicitation but suggest that other MAMPs can elicit MTI.
A. Higher inoculum enhances ROS elicitation by Pf0-1 and Pf0-1(pCPP6225) (pT3SS) but does not rescue the loss of ROS elicitation by ΔfliC variants of these two strains.
B. Challenge inoculum effector translocation assays indicate that all Pf0-1 derivatives tested can elicit stronger functional MTI at the higher level of inoculum.
C. Challenge inoculum HR assays indicate that all Pf0-1 derivatives tested can elicit stronger functional MTI at the higher level of inoculum. The assays and analyses for ROS, challenge effector translocation, and HR suppression were performed as described in Fig. 1.
Fig. S4. Colocalization of SP–FliCPf0-1–YFP–HA and the plasma membrane marker pm-rk (PM–mCherry) in plasmolysed N. benthamiana epidermal cells shows association of SP–FliCPf0-1 with the plasma membrane. A. tumefaciens GV3101 strains carrying binary vectors expressing SP–FliCPf0-1–YFP–HA and AtPIP2A–mCherry (PM–mCherry) were co-infiltrated into N. benthamiana leaves as described in Fig. 3. Plasmolysis, with 700 mM sucrose, of excised, infiltrated leaf regions was then used to enhance analysis of the distribution of SP–FliCPf0-1–YFP–HA in epidermal cells. Both unplasmolysed and plasmolysed cells show substantial colocalization of SP–FliCPf0-1–YFP–HA with the plasma membrane marker, but plasmolysed cells show no evidence of SP–FliCPf0-1–YFP–HA in the apoplast. Scale bar = 30 μm.
Fig. S5. Agrobacterium-mediated transient expression of FliCPtoDC3000-HA in N. benthamiana, tobacco and tomato leaves does not elicit death-associated tissue collapse.
A. Production in the cytosol of the test plants of FliCPtoDC3000 with only a C-terminal HA tag results in the same lack of a death response as was observed with FliCPtoDC3000–YFP–HA. Inoculations and photography were as described for Figs 3 and 5. Photographs were taken at 5 days post inoculation.
B. Immunoblot with HA antibody confirms expression of FliCPf0-1-HA in plant leaves.
Table S1. Strains and plasmids used in this study.
Table S2. Oligonucleotide primers used in this study.
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