Stringent response regulators (p)ppGpp and DksA positively regulate virulence and host adaptation of Xanthomonas citri

Abstract

SummaryThe bacterial stringent response is a response to nutrition deprivation and other stress conditions. In Gram‐negative bacteria, this process is mediated by the small signal molecules guanosine pentaphosphate pppGpp and guanosine tetraphosphate ppGpp (collectively referred to as (p)ppGpp), and the RNA polymerase‐binding transcription factor DksA. The (p)ppGpp synthetase RelA and the bifunctional (p)ppGpp synthase/hydrolase SpoT are responsible for cellular (p)ppGpp levels. Here, we investigated the roles of DksA and (p)ppGpp in the virulence traits of Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker. ΔdksA and (p)ppGpp‐deficient ΔspoTΔrelA strains caused reduced virulence and compromised growth in host plants, indicating that DksA and (p)ppGpp are required for full virulence of Xcc. To characterize the effect of stringent response regulators on gene expression, RNA‐seq was conducted using ΔdksA and ΔspoTΔrelA mutant strains grown in hrp‐inducing XVM2 medium. Transcriptome analyses showed that DksA and (p)ppGpp repressed the expression of genes encoding tRNAs, ribosome proteins, iron acquisition and flagellum assembly, and enhanced the expression of genes for histidine metabolism, type 3 secretion system (T3SS), type 2 secretion system (T2SS) and TonB‐dependent transporters. Phenotypically, the ΔdksA and ΔspoTΔrelA strains displayed altered motility, enhanced siderophore production and were unable to cause the hypersensitive response on non‐host plants. In conclusion, stringent response regulators DksA and (p)ppGpp play an important role in virulence, nutrition uptake and host adaptation of Xcc.

The HrpG/HrpX Regulon of Xanthomonads—An Insight to the Complexity of Regulation of Virulence Traits in Phytopathogenic Bacteria

“…Stringent response in bacteria is induced by stress or metabolite starvation [ 73 ] and is mediated by internal levels of guanosine pentaphosphate [(p)ppGpp], which is synthesized and degraded by RelA and SpoT [ 73 ]. It was recently reported that deletion of relA and spoT homologs in Xcci completely abolished pathogenicity in citrus and caused a significant reduction in the expression of hrpG, hrpX, and their downstream targets [ 74 ]. Similar phenotypes were observed upon deletion of transcriptional regulator dksA [ 74 ], which is associated with alteration of the affinity of the RNA polymerase to (p)ppGpp [ 75 ] ( Figure 2 ).…”

“…It was recently reported that deletion of relA and spoT homologs in Xcci completely abolished pathogenicity in citrus and caused a significant reduction in the expression of hrpG, hrpX, and their downstream targets [ 74 ]. Similar phenotypes were observed upon deletion of transcriptional regulator dksA [ 74 ], which is associated with alteration of the affinity of the RNA polymerase to (p)ppGpp [ 75 ] ( Figure 2 ). These observations suggest that hrp gene expression is regulated by internal levels of (p)ppGpp that are presumably controlled by nutrient limitation.…”

“…Expression, stability, and activity of HrpG and HrpX are dependent on numerous regulatory factors. HrpG and HrpX are negatively or positively controlled by two-component system kinases and response regulators [ 60 , 62 ], ATPases [ 102 ], transcriptional regulators [ 53 , 63 , 77 , 81 , 84 , 91 , 93 , 103 , 104 , 105 ], small signaling molecules [ 74 , 101 ] and histone-like nucleoid-structuring (H-NS) protein [ 106 , 107 , 108 ] ( Figure 2 ). However, most studies do not demonstrate direct interactions with HrpG, HrpX, or their promoters and it appears that the regulatory function on this regulon by most of these factors is probably indirect.…”

mCherry fusions enable the subcellular localization of periplasmic and cytoplasmic proteins in Xanthomonas sp.

“…Cell cultures were distributed in the wells of a 24-wells microtiter plate and incubated in a microtiter plate reader (Synergy H1M1; BioTek) at 30˚C with constant agitation (200 rpm) and OD 600 nm were measured every 30 min for 20 hours [24]. The in planta growth curves were performed according to procedures outlined by Zhang et al [25], with modifications. The bacterial cultures were adjusted to 10 6 CFU/mL with sterile 10 mM MgCl 2 solution and infiltrated into the abaxial surface leaves of Valencia sweet orange (Citrus sinensis) using a needleless syringe.…”

Application of TonB-Dependent Transporters in Vaccine Development of Gram-Negative Bacteria

“…However, under iron limiting conditions (for example, in vivo ), Fur cannot bind the sequence of the Fur box resulting in derepression of genes that encode siderophore transporters (including TBDTs), proteins involved in siderophore biosynthesis and iron metabolism ( Figure 2 ) ( Lee and Helmann, 2007 ; Noinaj et al., 2010 ). In addition to Fur, multiple regulatory factors including small RNAs, ChvR ( Frohlich et al., 2018 ), OmrA and OmrB ( Guillier and Gottesman, 2008 ), string response regulators (P) ppGpp and DksA ( Zhang et al., 2019 ), participate in the regulation of TBDTs for siderophore.…”