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Lecture 2

Lecture 2 Summary

Cell and Systems Biology
Course Code
Darrel Desveaux

of 2
Type III Secretion System
TTSS in gram-negative bacteria is needle like with long pilus similar to flagella.
HrpA forms pilus via spontaneous polymerization. HrcN is ATPase that powers translocation.
Pto HrcC with non-functional TTSS grows less.
Hrp = hypersensitive reaction and pathogenecity genes. Hrc = Hrp conserved genes.
Hrc Gene System
Exchangeable effector locus – Hrc gene cluster – conserved effector locus.
Hrc gene cluster is conserved core operon with HrcL regulatory gene in Pathogenicity Island.
Virulent genes only present in virulent strains. Different CG content occupying large chromosomal
regions. Flanked by direct repeats or bordered by tRNA or mobile elements.
Instability indicative of horizontal gene transfer.
HrcL is master TF: with signal, protease stop degrading HrcR. HrcR + Q54 + HrcS transcribe HrcL.
Shotgun Screen to Identify TTSE
Identified 1 factor that cause HR in soybeans.
Grow P. syringae pv glycinea and prepare shredded gDNA and transform gDNA into E.coli colonies.
Triparental mating: donor with Psg6DNA + Helper E.coli + recipient with Psg5 lacking Avr gene.
Grow Psg5 transconjugates and inoculate on plant leaf to assess HR. If fragment in donor contained
TTSE Avr gene then recipient should cause HR.
Bioinformatic Screen for TTSE
All TTSE contain Secretion Signal + catalytic/binding domain with optional chaperone binding site.
Look for Hrp Box (cis element bound by HrcL) upstream of ORF in complete genome of DC3000.
Look for ORF with characterisitic SS at N’ of TTSE.
Found 22 Pto TTSE but this is not a functional screen for translocation.
Transposon Screen for TTSE
AvrRpt2 is TTSE that cuase HR. without SS it does not induce HR.
Chimeric SS of AvrRpm1 + AvrRpt2 catalytic domain cause HR, but SS of AvrRpm1 long does not.
Create mini-transposon of AvrRpt2N (SS) and it will randomly integrate into plant genome.
Now put random gDNA in front of AvrRpt2, if there is HR, then AvrRpt2N (SS) must be after a SS
and is successfully translocated into the nucleus.
Go back and sequence fragment. Identified 13 effectors and confirmed translocation.
But laborious and there is bias of transposon insertion.
FACS screen
Functional screen using fluorescence activated cell sorting.
Make 2 constructs: arabinose promoter – HrpL + gDNA – AvrRpt2N (SS) – GFP.
Look for HrpL inducible clone that only grow when arabinose is applied and appear fluorescent.
If fluorescent, gDNA must contain HrpBox promoter, typical of TTSE. Now can clone gene.
Found 29 pto effectors and confirmed translocation. Also 19 bean TTSE.
HrpA- mutant lacks pilus and induce strong callose, PTI in both WT and NahG plants.
DC3000 induce little PTI as TTSE suppress PTI defence.
Mutant HrcC expressed in plant induce strong PTI, but WT HrcC transgenic expression in plant reduced
PTI and DC3118 grew better showing TTSE blocks basal PAMP.
CEL (conserved effector locus) bacteria caused more callose PTI in WT than NahG, as TTSE outside
of CEL may have blocked PTI.
PTI is SA-dependent.