Characterization of the
hrpC
and
hrpRS
Operons of
Pseudomonas syringae
Pathovars Syringae, Tomato, and Glycinea and Analysis of the Ability of
hrpF
,
hrpG
,
hrcC
,
hrpT
, and
hrpV
Mutants To Elicit the Hypersensitive Response and Disease in Plants
Authors: Deng Wen-Ling , Preston Gail , Collmer Alan , Chang Chun-Jung , Huang Hsiou-Chen 
Published: December 2019

ABSTRACT

The species
Pseudomonas syringae
encompasses plant pathogens with differing host specificities and corresponding pathovar designations.
P. syringae
requires the Hrp (type III protein secretion) system, encoded by a 25-kb cluster of
hrp
and
hrc
genes, in order to elicit the hypersensitive response (HR) in nonhosts or to be pathogenic in hosts. DNA sequence analysis of the
hrpC
and
hrpRS
operons of
P. syringae
pv. syringae 61 (brown spot of beans),
P. syringae
pv. glycinea U1 (bacterial blight of soybeans), and
P. syringae
pv. tomato DC3000 (bacterial speck of tomatos) revealed that the 13 genes comprising the right half of the
hrp
cluster (including those in the previously sequenced
hrpZ
operon) are conserved and identically arranged. The
hrpC
operon is comprised of
hrpF
,
hrpG
,
hrcC
,
hrpT
, and
hrpV. hrcC
encodes a putative outer membrane protein that is conserved in all type III secretion systems. The other four genes appear to be characteristic of group I Hrp systems, such as those possessed by
P. syringae
and
Erwinia amylovora
. The predicted products of these four genes in
P. syringae
pv. syringae 61 are HrpF (8 kDa), HrpG (15.4 kDa), HrpT (7.5 kDa), and HrpV (13.4 kDa). HrpT is a putative outer membrane lipoprotein. HrpF, HrpG, and HrpV are all hydrophilic proteins lacking N-terminal signal peptides. The HrpG, HrcC, HrpT, and HrpV proteins of
P. syringae
pathovars syringae and tomato (the two most divergent pathovars) had at least 76% amino acid identity with each other, whereas the HrpF proteins of these two pathovars had only 36% amino acid identity. The HrpF proteins of
P. syringae
pathovars syringae and glycinea also showed significant similarity to the HrpA pilin protein of
P. syringae
pathovar tomato. Functionally nonpolar mutations were introduced into each of the genes in the
hrpC
operon of
P. syringae
pv. syringae 61 by insertion of an
nptII
cartridge lacking a transcription terminator. The mutants were assayed for their ability to elicit the HR in nonhost tobacco leaves or to multiply and cause disease in host bean leaves. Mutations in
hrpF
,
hrcC
, and
hrpT
abolished or greatly reduced the ability of
P. syringae
pv. syringae 61 to elicit the HR in tobacco. The
hrpG
mutant had only weakly reduced HR activity, and the activity of the
hrpV
mutant was indistinguishable from that of the wild type. Each of the mutations could be complemented, but surprisingly, the
hrpV
subclone caused a reduction in the HR elicitation ability of the Δ
hrpV
::
nptII
mutant. The
hrpF
and
hrcC
mutants caused no disease in beans, whereas the
hrpG
,
hrpT
, and
hrpV
mutants had reduced virulence. Similarly, the
hrcC
mutant grew little in beans, whereas the other mutants grew to intermediate levels in comparison with the wild type. These results indicate that HrpC and HrpF have essential functions in the Hrp system, that HrpG and HrpT contribute quantitatively but are not essential, and that HrpV is a candidate negative regulator of the Hrp system.