Table 1.
Functional Categories of Genes Positively Regulated by HrpG Belonging Either to the HrpB-Dependent or to the HrpB-Independent Pathway
Figure 1.
The HrpG Regulator Affects Virulence and Host Colonisation Factors in an hrpB-Independent Manner
Enzymatic activities for (A) endoglucanase, (B) polygalacturonase, and (C) catalase, as well as (D) production of EPS and (E) efe gene expression, are presented. Biological tests were performed in an HrpG-deficient strain (ΔhrpG) and the wild-type GMI1000 strain (wild-type), both bearing the empty vector pL, as well as in the wild-type strain bearing plasmid pLG, which overexpresses HrpG. An hrpB mutant overexpressing HrpG (hrpB::Ω/pLG) was also included in this set of experiments to check the influence of HrpB in the regulation. Measured values with standard deviations are presented below quantitative tests [units: (A, B): halo surface in cm2, (C) foam height in cm, (E) Miller units].
Table 2.
A Subset of Genes with Known Function Whose Expression Is Dependent on HrpG but Not on HrpB, including Several Known or Candidate Pathogenicity Functions
Figure 2.
HrpG Promotes Phytohormone Production in R. solanacearum
(A) Auxin production by a strain deleted for hrpG (ΔhrpG), the GMI1000 reference strain (wild-type), an HrpG overproducer (wild-type/pLG), and a strain overproducing the regulator in an hrpB mutant background (hrpB::Ω/pLG). Auxin concentrations in culture medium of overnight-grown cultures are presented as OD530/OD600 ratios.
(B) Ethylene production by wild-type R. solanacearum and an ethylene-forming enzyme mutant (efe) bearing the pL empty vector or its hrpG-overexpressing derivative (pLG). The hormone was measured by chromatography from the gas phase of overnight-sealed cultures.
(C) Triple-response assay with bacterial ethylene. Gas phases of cultures equivalent to those in (B) were injected onto sealed flasks containing A. thaliana seedlings photographed after 96 h of growth in the dark. The extent of the triple-response symptoms (cotyledon curvature, hypocotyl shortening, and root growth inhibition) is correlated to ethylene production.
Figure 3.
Effects of Ethylene Produced by R. solanacearum on the Plant Host
Expression of A. thaliana ethylene-responsive genes upon inoculation with GMI1000. mRNA levels of the ethylene response factor 1 gene (ERF1, hollow symbols and dotted lines) and the pathogen response gene 4 (PR4, shaded symbols) were measured by quantitative real-time PCR during infection by a wild-type (triangles) or an ethylene-deficient strain (squares).
Figure 4.
Influence of the hrpG-Regulated Functions Belonging to the TTSS-Independent Pathway on R. solanacearum Pathogenicity
Pathogenicity tests on tomato plants. In the Pkan::hrpB (triangles) and Ptac::hrpB (squares) strains, transcription of the TTSS is constitutive and uncoupled to HrpG. These strains (solid symbols) and their ΔhrpG counterparts (hollow symbols and dotted lines) were used to inoculate tomato plants, as well as the wild-type strain GMI1000 (circles). The percentage of wilting recorded upon time is represented. In all cases, points indicate mean values and error bars indicate standard deviations of at least three experiments.
Figure 5.
Schematic Model of the Hrp Regulation Cascade in R. solanacearum
A plant signal is sensed by the outer-membrane receptor PrhA and is transduced to PrhJ through PrhI/R [63]. HrpG is the key/central component that integrates in the cascade at least three signals, as its activity depends on the plant host contact [16], bacterial metabolic signals related to growth conditions [17], and a phc-dependent quorum sensing signal [18]. HrpG regulates the expression of the TTSS apparatus and effector genes (through HrpB activation), and it controls as well the newly described HrpB-independent functions that also contribute to pathogenicity. Rounded forms symbolise proteins, and open arrows, gene sequences.