Quorum Sensing

we recommend performing preliminary tests to examine the

stability of thegfp-plasmid and fluorescence emission of the

tested bacterial strains in vitro and in planta.

4. It is important to provide good aeration conditions to assure
   fast growing of the bacterial cultures.


5. We recommend using no more than three discs per plate to
   avoid diffusion of the DSF among discs.


6. Black microtiter plates reduce background and cross-reactivity
   between samples.


7. Here we present an example to determine disease severity in
   pelargonium plants based on a four-grade scale: (1) no symp-
   toms, plants look healthy as mock-inoculated plants; (2) low
   severity, small necrotic areas are observed on one or two leaves
   close to the inoculation site; (3) moderate severity, more than
   three leaves develop necrotic areas; and (4) high severity, wilt-
   ing, and necrosis symptoms are observed on most leaves or the
   plant died.


8. Depend on the pathogen–host system colonization can be
   determined at different time intervals after inoculation and
   samples can be taken from different plant tissues.


9. Aggregate formation is variable depending on the bacteria and
   growth conditions, therefore it is recommended to perform
   this assay at different time of incubation.

Acknowledgement

We are grateful to Steven Lindow for providing strainXanthomonas

campestrispv.campestris8523 (pKLN55). Contribution from the

Agricultural Research Organization, The Volcani Center, Bet

Dagan, Israel.

References

1. Barber CE, Tang JL, Feng JX, Pan MQ, Wilson
   TJG, Slater H et al (1997) A novel regulatory
   system required for pathogenicity ofXantho-
   monas campestrisis mediated by a small diffus-
   ible signal molecule. Mol Microbiol
   24:555–566


2. Rai R, Ranjan M, Pradhan BB, Chatterjee S
   (2012) Atypical regulation of virulence-
   associated functions by a diffusible signal factor
   inXanthomonas oryzaepv.oryzae. Mol Plant
   Microbe Interact 25:708–801


3. von Bodman SB, Bauer WD, Coplin DL
   (2003) Quorum sensing in plant-pathogenic
   bacteria. Annu Rev Phytopathol 41:455–482
   4. Ryan RP, Dow JM (2011) Communication
   with growing family: diffusible signal factor
   (DSF) signaling in bacteria. Trends Microbiol
   19:145–152
   5. Wang LH, He Y, Gao Y, JE W, Dong Y, He C
   et al (2004) A bacterial cell–cell communica-
   tion signal with cross-kingdom structural ana-
   logues. Mol Microbiol 51:903–912
   6. Bi H, Christensen QH, Feng Y, Wang H, Cro-
   nan JE (2012) TheBurkholderia cenocepacia
   BDSF quorum sensing fatty acid is synthesized
   by a bifunctional crotonase homologue having
   both dehydratase and thioesterase activities.
   Mol Microbiol 83:840–855

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