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Regulation Of Virulence In The Plant Pathogen Pseudomonas Syringae Pv. Tomato Dc3000

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The  type  III  secretion  system  (T3SS)  is  required  for  virulence  of  the  gram-­- negative   plant   pathogen   Pseudomonas  syringae   pv.   tomato  DC3000   (DC3000)   in   tomato   and   Arabidopsis.   The   alternative   sigma   factor   HrpL   directly   regulates   expression  of  T3SS  genes  by  binding  to  a  short  DNA  sequence  designated  as  the   "hrp  promoter".  The  ability  of  DC3000  to  colonize  plants,  subdue  multiple  layers   of  plant  defense  and  multiply  in  plant  tissues  relies  on  the  activities  carried  out   by  the  many  T3SS  regulon  members  (known  collectively  as  hrp  genes).     Efforts   to   identify   genes   involved   in   pathogenicity   were   initiated   over   three   decades   ago.   However,   HrpL   binding   to   hrp   promoters   has   never   been   directly   demonstrated   and   it   is   unclear   if   the   list   of   HrpL-­-regulated   genes   is   complete.   The   first   goal   of   the   research   described   here   was   to   systemically   and   exhaustively  identify  HrpL-­-binding  sites  and  likely  hrp  promoters  in  the  DC3000   genome.   Employing   chromatin   immuno-­-precipitation,   coupled   with   high-­- throughput   sequencing   (ChIP-­-Seq)   and   transcription   start   site   analysis   (modified   RNA-­-Seq),   we   found   twenty   sites   representing   novel   hrp   promoters.   Using   deletion   analysis,   we   attempted   to   determine   if   the   genes   downstream   from   a   subset   of   these   promoters   could   be   linked   to   virulence.   However,   the   deletions   did   not   affect   the   hypersensitive   response   or   in   planta   growth   of   the   resulting   strains.  Interestingly,  many  new  HrpL  regulon  members  appear  to  be  unrelated   to  the  T3SS  (based  on  their  annotations),  and  orthologs  for  some  of  these  can  be   identified   in   non-­-pathogenic  bacteria.   The   connection  of  these  new  HrpL  regulon   members  to  virulence  is  not  obvious.   The   HrpL   regulon   is   activated   as   a   result   of   a   chain   of   events,   most   of   which   are   not   well   understood.   It   is   known   that   RpoN,   which   controls   the   transcription   of   hrpL   in   DC3000,   is   required   for   virulence   in   several   bacterial   species.   Motivated   by   the   hypothesis   that   genes   are   coordinately   regulated   in   order   to   serve   a   strategic   purpose   (e.g.,   virulence),   our   second   goal   was   to   look   for   other   genes   activated   by   RpoN   in   parallel   with   hrpL.   RpoN   ([sigma]54)   requires   specialized   enhancer-­-binding   proteins   (EBPs)   in   order   to   activate   transcription.   This   arrangement   presumably   allows   the   cell   to   respond   to   environmental   signals   by   modifying   the   transcription   of   particular   genes.   Using   ChIP-­-Seq   and   RNA-­-Seq,   we   identified   candidate   RpoN-­-dependent   genes   as   well   as   genes   that   were   differentially   expressed   under   hrp-­-inducing   conditions.   This   initial   survey   includes   more   than   200   likely   RpoN-­-regulated   genes   involved   in   flagella   biosynthesis,   energy   metabolism,   nitrogen   metabolism,   transport   and   binding   proteins,  and  small  noncoding  RNAs,  as  well  as  putative  regulatory  proteins  and   EBPs.  Among  the  genes  that  were  differentially  regulated  between   hrp-­-inducing   and   repressing   conditions,   more   than   one   dozen   appear   to   be   regulated   by   RpoN   and  are  therefore  potentially  important  in  functions  related  to  plant  association   or  virulence.

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2014-08-18

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Pseudomonas syringae; Type III secretion; virulence; sigma factor; enhancer; HrpL; RpoN; regulation; transcription

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Union Local

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Cartinhour, Samuel

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Myers, Christopher R
Perry, Keith Lloyd
Helmann, John D
Collmer, Alan Raymond

Degree Discipline

Plant Pathology

Degree Name

Ph. D., Plant Pathology

Degree Level

Doctor of Philosophy

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Government Document

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dissertation or thesis

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