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CSB452 LECTURE 2
Type III Secretion System
๎€
TTSS in gram-negative bacteria is needle like with long pilus similar to f lagella.
๎€
HrpA forms pilus via spontaneous polymerization. HrcN is ATPase that powers translocation.
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Pto HrcC
with non-functional TTSS grows less.
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Hrp = hypers ensitive reaction and pathogenecity genes. H rc = Hrp conserved genes.
Hrc Gene System
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Exchangeable effector locus โ€“ Hrc gene cluster โ€“ conserved effector locus.
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Hrc gene cluster is conserved core operon with HrcL regulatory gene in Pathogenicity Island.
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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.
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Instability indicative of horizont al gene transfer.
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HrcL is master TF: with signal, protease stop degrading HrcR. HrcR + Q
54
+ HrcS transcribe HrcL.
Shotgun Screen to Identify TTSE
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Identified 1 factor that cause HR in soybeans.
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Grow
P. syringae
pv glycinea and prepare shredded gDNA and transform gDNA into
E.coli
colonies.
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Triparental mating: donor with Psg6DNA + Helper
E.coli
+ recipient with Psg5 lacking Avr gene.
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Grow Psg5 transconjugates and inoculate on plant leaf to assess HR. If fragment in donor contained
TTSE Avr gene then recipient should cause HR.
Bioinfor matic Screen for TTSE
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All TTSE contain Secretion Signal + catalytic/binding domain with optional chaperone binding site.
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Look for Hrp Box (
cis
element bound by HrcL) upstream of ORF in complete genome of DC3000.
๎€
Look for ORF w ith characterisitic SS at Nโ€™ of TTSE.
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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.
1
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Document Summary

Ttss in gram-negative bacteria is needle like with long pilus similar to flagella. hrp = hypersensitive reaction and pathogenecity genes. 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. Flanked by direct repeats or bordered by trna or mobile elements. hrcl is master tf: with signal, protease stop degrading hrcr. 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. Ttse avr gene then recipient should cause hr. all ttse contain secretion signal + catalytic/binding domain with optional chaperone binding site.

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