Disrup[on of Bacterial Endoparasi[sm of Plant Roots Using Butyric Acid

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Poa ampla seeds sterilized with 30 min with 4%. NaOCl. • Seeds placed on agarose media containing 0, 0.5, or. 1.0 mM of butyric acid. • Seeds inoculated with ...
Disrup'on  of  Bacterial   Endoparasi'sm  of  Plant  Roots  Using   Butyric  Acid     James  White  and  Kathryn  Kingsley   Dept.  Plant  Biology   Rutgers  University   [email protected]  

Methodology   •  Poa  ampla  seeds  sterilized  with  30  min  with  4%   NaOCl.     •  Seeds  placed  on  agarose  media  containing  0,  0.5,  or   1.0  mM  of  butyric  acid.   •  Seeds  inoculated  with  endoparasi'c/endophy'c   bacterium  Pseudomonas  fluorescens.   •  ASer  several  days  seedlings  were  stained  overnight   with  reac've  oxygen  stain  diaminobenzidine  (DAB),   then  counterstained  with  aniline  blue  to  visualize   bacteria  within  plant  root  hairs.  

Root  'p  showing  long  hairs  (arrows)  in  a  root  that  was   not  treated  with  butyric  acid.  

0.5  mM  butyric  acid  treatment  showing  shorter  root  hairs  due  to   fewer  bacteria  entering  the  root  cells  at  the  'p  meristem  (blue   arrow).  

Note  bacterial  biofilm    around  meristem  

1  mM  butyric  acid  treatment  showing  total  absence  of  root  hairs   due   to   cessa'on   of   intracellular   invasion   by   bacteria.   Without   intracellular  bacteria  no  root  hairs  form.  

Root   hair   from   treatment   without   butyric   acid   showing   abundant   intracellular   bacteria.   Arrows   show   intracellular  bacteria.  Bacteria  s'mulate  root  hair  development  through  produc'on  of  auxin.  The  bacteria  exit   root   hairs   at   the   'p   where   the   cell   wall   is   thin.   The   slight   reddish   colora'on   indicates   presence   of   reac've   oxygen  around  intracellular  bacteria  due  to  host  defensive  reac'on.    

Root   hair   from   treatment   without   butyric   acid   showing   two   bacteria   (black  arrow)  exi'ng  the  hair  at  the  'p  of  the  hair  where  the  cell  wall  is   thin.     Bacteria   are   present   in   the   root   cells   as   L-­‐forms   (blue   arrows).   The  exi'ng  bacteria  will  reform  cell  walls  to  become  rods.  

Root   hair   from   0.5   mM   butyric   acid   treatment.   Intracellular   bacteria   are   sparse   in   this   treatment.   Most   of   the   bacteria   have   already   exited   the   hair.   No   L-­‐forms   are   visible   in   this  hair.  Due  to  presence  of  butyric  acid  in  the  medium  few  bacteria  entered  the  root   cells   at   the   growing   root   meristem.   The   few   bacteria   that   did   enter   cells   s'mulated   root  hair  forma'on  but  because  their  number  was  few,  root  hair  length  growth  was   restricted.  

Another   root   hair   from   the   0.5   mM   butyric   acid   treatment.   All   intracellular  bacteria  have  already  exited  the  hair.    

Another   hair   from   the   0.5   mM   butyric   acid   treatment  with  few  if  any  L-­‐forms  in  the  hair.    

Hair  from  the  0.5  mM  butyric  acid  treatment  showing  an  exi'ng   bacterium  at  the  'p  (arrow).    Three  bacteria  are  present  within   the  hair  below  the  exi'ng  bacterium.  

Conclusions   •  Butyric  acid  reduced  (0.5  mM  level)  and  completely   eliminated  (1  mM  level)  conversion  of  Pseudomonas   fluorescens  from  the  free-­‐living  state  to  the   endoparasi'c.   •  The  quan'ty  of  bacteria  that  enter  into  root  'p   meristem  cells  directly  correlates  with  the  length  of   root  hairs.  A  greater  number  of  bacteria  that  enter  at   the  root  meristem  results  in  longer  root  hair  growth.