Histidine Phosphotransfer Research Articles

Two-component signaling system in plants: interaction network and specificity in response to stress and hormones.

Plants are exposed to various environmental challenges that can hamper their growth, development, and productivity. Being sedentary, plants cannot escape from these unfavorable environmental conditions and have evolved various signaling cascades to endure them. The two-component signaling (TCS) system is one such essential signaling circuitry present in plants regulating responses against multiple abiotic and biotic stresses. It is among the most ancient and evolutionary conserved signaling pathways in plants, which include membrane-bound histidine kinases (HKs), cytoplasmic histidine phosphotransfer proteins (Hpts), and nuclear or cytoplasmic response regulators (RRs). At the same time, TCS also involved in many signaling circuitries operative in plants in response to diverse hormones. These plant growth hormones play a significant role in diverse physiological and developmental processes, and their contribution to plant stress responses is coming up in a big way. Therefore, it is intriguing to know how TCS and various plant growth regulators, along with the key transcription factors, directly or indirectly control the responses of plants towards diverse stresses. The present review attempts to explore this relationship, hoping that this knowledge will contribute towards developing crop plants with enhanced climate resilience.

Cytokinins Stimulate Plasmodesmatal Transport in Leaves.

Plant cells are connected by plasmodesmata (PD), nanoscopic channels in cell walls that allow diverse cytosolic molecules to move between neighboring cells. PD transport is tightly coordinated with physiology and development, although the range of signaling pathways that influence PD transport has not been comprehensively defined. Several plant hormones, including salicylic acid (SA) and auxin, are known to regulate PD transport, but the effects of other hormones have not been established. In this study, we provide evidence that cytokinins promote PD transport in leaves. Using a green fluorescent protein (GFP) movement assay in the epidermis of Nicotiana benthamiana, we have shown that PD transport significantly increases when leaves are supplied with exogenous cytokinins at physiologically relevant concentrations or when a positive regulator of cytokinin responses, ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 5 (AHP5), is overexpressed. We then demonstrated that silencing cytokinin receptors, ARABIDOPSIS HISTIDINE KINASE 3 (AHK3) or AHK4 or overexpressing a negative regulator of cytokinin signaling, AAHP6, significantly decreases PD transport. These results are supported by transcriptomic analysis of mutants with increased PD transport (ise1–4), which show signs of enhanced cytokinin signaling. We concluded that cytokinins contribute to dynamic changes in PD transport in plants, which will have implications in several aspects of plant biology, including meristem patterning and development, regulation of the sink-to-source transition, and phytohormone crosstalk.

Function of the pseudo phosphotransfer proteins has diverged between rice and Arabidopsis.

The phytohormone cytokinin plays a significant role in nearly all aspects of plant growth and development. Cytokinin signaling has primarily been studied in the dicot model Arabidopsis, with relatively little work done in monocots, which include rice (Oryza sativa) and other cereals of agronomic importance. The cytokinin signaling pathway is a phosphorelay comprised of the histidine kinase receptors, the authentic histidine phosphotransfer proteins (AHPs) and type-B response regulators (RRs). Two negative regulators of cytokinin signaling have been identified: the type-A RRs, which are cytokinin primary response genes, and the pseudo histidine phosphotransfer proteins (PHPs), which lack the His residue required for phosphorelay. Here, we describe the role of the rice PHP genes. Phylogenic analysis indicates that the PHPs are generally first found in the genomes of gymnosperms and that they arose independently in monocots and dicots. Consistent with this, the three rice PHPs fail to complement an Arabidopsis php mutant (aphp1/ahp6). Disruption of the three rice PHPs results in a molecular phenotype consistent with these elements acting as negative regulators of cytokinin signaling, including the induction of a number of type-A RR and cytokinin oxidase genes. The triple php mutant affects multiple aspects of rice growth and development, including shoot morphology, panicle architecture, and seed fill. In contrast to Arabidopsis, disruption of the rice PHPs does not affect root vascular patterning, suggesting that while many aspects of key signaling networks are conserved between monocots and dicots, the roles of at least some cytokinin signaling elements are distinct.

Genome-Wide Characterization and Expression of Two-Component System Genes in Cytokinin-Regulated Gall Formation in Zizania latifolia.

The thickening of Zizania latifolia shoots, referred to as gall formation, depends on infection with the fungal endophyte Ustilago esculenta. The swollen and juicy shoots are a popular vegetable in Asia. A key role for cytokinin action in this process was postulated. Here, trans-zeatin stimulated swelling in fungi-infected Z. latifolia. A two-component system (TCS) linked cytokinin binding to receptors with transcriptional regulation in the nucleus and played important roles in diverse biological processes. We characterized 69 TCS genes encoding for 25 histidine kinase/histidine-kinase-like (HK(L)) (21 HKs and 4 HKLs), 8 histidine phosphotransfer proteins (HP) (5 authentic and 3 pseudo), and 36 response regulators (RR; 14 type A, 14 type B, 2 type C, and 6 pseudo) in the genome of Z. latifolia. These TCS genes have a close phylogenetic relationship with their rice counterparts. Nineteen duplicated TCS gene pairs were found and the ratio of nonsynonymous to synonymous mutations indicated that a strong purifying selection acted on these duplicated genes, leading to few mutations during evolution. Finally, ZlCHK1, ZlRRA5, ZIRRA9, ZlRRA10, ZlPRR1, and ZlPHYA expression was associated with gall formation. Among them, ARR5, ARR9, and ZlPHYA are quickly induced by trans-zeatin, suggesting a role for cytokinin signaling in shoot swelling of Z. latifolia.

The pre-mRNA splicing factor RDM16 regulates root stem cell maintenance in Arabidopsis.

Pre-mRNA (messenger RNA) splicing participates in the regulation of numerous biological processes in plants. For example, alternative splicing shapes transcriptomic responses to abiotic and biotic stress, and controls developmental programs. However, no study has revealed a role for splicing in maintaining the root stem cell niche. Here, a screen for defects in root growth in Arabidopsis thaliana identified an ethyl methane sulfonate mutant defective in pre-mRNA splicing (rdm16-4). The rdm16-4 mutant displays a short-root phenotype resulting from fewer cells in the root apical meristem. The PLETHORA1 (PLT1) and PLT2 transcription factor genes are important for root development and were alternatively spliced in rdm16-4 mutants, resulting in a disordered root stem cell niche and retarded root growth. The root cap of rdm16-4 contained reduced levels of cytokinins, which promote differentiation in the developing root. This reduction was associated with the alternative splicing of genes encoding cytokinin signaling factors, such as ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN5 and ARABIDOPSIS RESPONSE REGULATORS (ARR1, ARR2, and ARR11). Furthermore, expression of the full-length coding sequence of ARR1 or exogenous cytokinin application partially rescued the short-root phenotype of rdm16-4. This reveals that the RDM16-mediated alternative splicing of cytokinin signaling components contributes to root growth.

Root-derived trans-zeatin cytokinin protects Arabidopsis plants against photoperiod stress.

Recently, a novel type of abiotic stress caused by a prolongation of the light period-coined photoperiod stress-has been described in Arabidopsis. During the night after the prolongation of the light period, stress and cell death marker genes are induced. The next day, strongly stressed plants display a reduced photosynthetic efficiency and leaf cells eventually enter programmed cell death. The phytohormone cytokinin (CK) acts as a negative regulator of this photoperiod stress syndrome. In this study, we show that Arabidopsis wild-type plants increase the CK concentration in response to photoperiod stress. Analysis of cytokinin synthesis and transport mutants revealed that root-derived trans-zeatin (tZ)-type CKs protect against photoperiod stress. The CK signalling proteins ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 2 (AHP2), AHP3 and AHP5 and transcription factors ARABIDOPSIS RESPONSE REGULATOR 2 (ARR2), ARR10 and ARR12 are required for the protective activity of CK. Analysis of higher order B-type arr mutants suggested that a complex regulatory circuit exists in which the loss of ARR10 or ARR12 can rescue the arr2 phenotype. Together the results revealed the role of root-derived CK acting in the shoot through the two-component signalling system to protect from the negative consequences of strong photoperiod stress.

Regulating polymyxin resistance in Gram-negative bacteria: roles of two-component systems PhoPQ and PmrAB.

Polymyxins (polymyxin B and colistin) are last-line antibiotics against multidrug-resistant Gram-negative pathogens. Polymyxin resistance is increasing worldwide, with resistance most commonly regulated by two-component systems such as PmrAB and PhoPQ. This review discusses the regulatory mechanisms of PhoPQ and PmrAB in mediating polymyxin resistance, from receiving an external stimulus through to activation of genes responsible for lipid A modifications. By analyzing the reported nonsynonymous substitutions in each two-component system, we identified the domains that are critical for polymyxin resistance. Notably, for PmrB 71% of resistance-conferring nonsynonymous mutations occurred in the HAMP (present in histidine kinases, adenylate cyclases, methyl accepting proteins and phosphatase) linker and DHp (dimerization and histidine phosphotransfer) domains. These results enhance our understanding of the regulatory mechanisms underpinning polymyxin resistance and may assist with the development of new strategies to minimize resistance emergence.

Genome-wide characterization of two-component system (TCS) genes in melon (Cucumis melo L.)

To better understand cytokinin signaling in melon (Cucumis melo L.), one of the most important fruit crops in the Cucurbitaceae family, we identified and characterized melon two-component system (TCS) genes in this study. The results showed that there were 51 genes encoding putative TCS proteins in melon, and these TCS genes were classified into 3 subgroups, with 17 HK(L)s (histidine kinase/histidine-kinase like; 9 HKs and 8 HKLs), 9 HPs (histidine phosphotransfer proteins; 6 authentic and 3 pseudo), and 25 RRs (response regulators; 8 Type-A, 11 Type-B and 6 pseudo). The identity values of these cytokinin signaling proteins were revealed by analyzing their conserved motifs, domains and amino acid sequences. By analyzing TCS genes in different plant species, we found that melon HK(L)s, HPs and RRs had closer phylogenetic relationships with cucumber genes than with the genes of other plants, and the expansion of melon cytokinin signaling genes might be attributed to segmental duplication events. Analysis of the putative promoter regions (2-kb upstream regions of the start codon) revealed the enrichment of stress- and hormone-response cis-elements. The involvement of these putative TCS genes in melon cytokinin signaling was further supported by qRT-PCR data.

Conformational Transitions of Histidine Kinases using Molecular Dynamics

One of the main signal transduction mechanisms in bacteria are two-component systems (TCS), which are comprised of homodimeric sensor histidine kinase (HK) and response regulator (RR) proteins (1). Histidine kinases are multidomain, transmembrane proteins which function by detecting a stimulus and then propagate this signal across the domains via a series of conformational transitions ruled by unstable transient interactions (2), subsequently resulting in an appropriate cellular response. The dimerization histidine phosphotransfer (DHp) domain, contains a phosphorylatable conserved histidine residue. Secondly, the catalytic ATP-binding (CA) domain, binds ATP and hence provides the phosphoryl group necessary for phosphorylating the histidine residue in the DHp. Finally, the phosphoryl group from the histidine residue of the DHp of HK is transferred to an aspartate residue of the RR protein. We investigate this signal transduction using coarse-grained and all-atom molecular dynamics simulations. A specific focus are the conformational transitions between the active and inactive states of the conserved kinase core. In the active conformation, the ATP of CA and phosphorylatable histidine of DHp are in an ideal orientation for phosphoryl group transfer whereas in the inactive conformation, the two domains are too distant.

New Insight into HPts as Hubs in Poplar Cytokinin and Osmosensing Multistep Phosphorelays: Cytokinin Pathway Uses Specific HPts.

We have previously identified proteins in poplar which belong to an osmosensing (OS) signaling pathway, called a multistep phosphorelay (MSP). The MSP comprises histidine-aspartate kinases (HK), which act as membrane receptors; histidine phosphotransfer (HPt) proteins, which act as phosphorelay proteins; and response regulators (RR), some of which act as transcription factors. In this study, we identified the HK proteins homologous to the Arabidopsis cytokinin (CK) receptors, which are first partners in the poplar cytokinin MSP, and focused on specificity of these two MSPs (CK and OS), which seem to share the same pool of HPt proteins. Firstly, we isolated five CK HKs from poplar which are homologous to Arabidopsis AHK2, AHK3, and AHK4, namely, HK2, HK3a, HK3b, HK4a, HK4b. These HKs were shown to be functional kinases, as observed in a functional complementation of a yeast HK deleted strain. Moreover, one of these HKs, HK4a, was shown to have kinase activity dependent on the presence of CK. Exhaustive interaction tests between these five CK HKs and the 10 HPts characterized in poplar were performed using two-hybrid and BiFC experiments. The resulting partnership was compared to that previously identified between putative osmosensors HK1a/1b and HPt proteins. Finally, in planta coexpression analysis of genes encoding these potential partners revealed that almost all HPts are coexpressed with CK HKs in four different poplar organs. Overall, these results allowed us to unravel the common and specific partnerships existing between OS and CK MSP in Populus.

New Insights into Multistep-Phosphorelay (MSP)/ Two-Component System (TCS) Regulation: Are Plants and Bacteria that Different?

The Arabidopsis multistep-phosphorelay (MSP) is a signaling mechanism based on a phosphorelay that involves three different types of proteins: Histidine kinases, phosphotransfer proteins, and response regulators. Its bacterial equivalent, the two-component system (TCS), is the most predominant device for signal transduction in prokaryotes. The TCS has been extensively studied and is thus generally well-understood. In contrast, the MSP in plants was first described in 1993. Although great advances have been made, MSP is far from being completely comprehended. Focusing on the model organism Arabidopsis thaliana, this review summarized recent studies that have revealed many similarities with bacterial TCSs regarding how TCS/MSP signaling is regulated by protein phosphorylation and dephosphorylation, protein degradation, and dimerization. Thus, comparison with better-understood bacterial systems might be relevant for an improved study of the Arabidopsis MSP.

Insights revealed by the co-crystal structure of the Saccharomyces cerevisiae histidine phosphotransfer protein Ypd1 and the receiver domain of its downstream response regulator Ssk1.

Two‐component signaling systems are the primary means by which bacteria, archaea, and certain plants and fungi react to their environments. The model yeast, Saccharomyces cerevisiae, uses the Sln1 signaling pathway to respond to hyperosmotic stress. This pathway contains a hybrid histidine kinase (Sln1) that autophosphorylates and transfers a phosphoryl group to its own receiver domain (R1). The phosphoryl group is then transferred to a histidine phosphotransfer protein (Ypd1) that finally passes it to the receiver domain (R2) of a downstream response regulator (Ssk1). Under normal conditions, Ssk1 is constitutively and preferentially phosphorylated in the phosphorelay. Upon detecting hyperosmotic stress, Ssk1 rapidly dephosphorylates and activates the high‐osmolarity glycerol (HOG) pathway, initiating a response. Despite their distinct physiological roles, both Sln1 and Ssk1 bind to Ypd1 at a common docking site. Co‐crystal structures of response regulators in complex with their phosphorelay partners are scarce, leaving many mechanistic and structural details uncharacterized for systems like the Sln1 pathway. In this work, we present the co‐crystal structure of Ypd1 and a near wild‐type variant of the receiver domain of Ssk1 (Ssk1‐R2‐W638A) at a resolution of 2.80 Å. Our structural analyses of Ypd1‐receiver domain complexes, biochemical determination of binding affinities for Ssk1‐R2 variants, in silico free energy estimates, and sequence comparisons reveal distinctive electrostatic properties of the Ypd1/Ssk1‐R2‐W638A complex that may provide insight into the regulation of the Sln1 pathway as a function of dynamic osmolyte concentration.

Genome-wide identification and expression analysis of two component system genes in Cicer arietinum

The two-component system (TCS) plays an important role in signal transduction pathways, cytokinin signaling and stress resistance of prokaryotes and eukaryotes. It is comprised of three types of proteins in plants; histidine kinases (HKs), histidine phosphotransfer proteins (HPs) and response regulators (RRs). Chickpea (Cicer arietinum L.) is one of the most important legume crops worldwide with special economic value in semi-arid tropics. Availability of complete genome sequence of chickpea presents a valuable resource for comparative analysis among angiosperms. In current study, Arabidopsis thaliana and Oryza sativa were used as reference plant species for comparative genomics analysis with C. arietinum. A genome-wide computational survey enabled us to identify putative members of TCS protein family including 18HKs, 26 RRs (7 type-A, 7 type-B, 2 type C and 10 pseudo) and 7 HPs (5 true and 2pseudo) genes in chickpea. The predicted TCS genes displayed family specific intron/exon organization and were randomly distributed across all the eight chromosomes. Comparative phylogenetic and evolutionary analysis suggested a variable conservation of TCS genes in relation to mono/dicot model plants and segmental duplication was the principal route of expansion for this family in chickpea. The promoter regions of TCS genes exhibited several abiotic stress-related cis-elements indicating their involvement in abiotic stress response. The expression analysis of TCS genes demonstrated stress (drought, heat, osmotic and salt) specific differential expression. Current study provides insight into TCS genes in C. arietinum, which will be helpful for further functional analysis of these genes in response to different abiotic stresses.

Genome-wide associations and epistatic interactions for internode number, plant height, seed weight and seed yield in soybean

BackgroundBreeding programs benefit from information about marker-trait associations for many traits, whether the goal is to place those traits under active selection or to maintain them through background selection. Association studies are also important for identifying accessions bearing potentially useful alleles by characterizing marker-trait associations and allelic states across germplasm collections. This study reports the results of a genome-wide association study and evaluation of epistatic interactions for four agronomic and seed-related traits in soybean.ResultsUsing 419 diverse soybean accessions, together with genotyping data from the SoySNP50K Illumina Infinium BeadChip, we identified marker-trait associations for internode number (IN), plant height (PH), seed weight (SW), and seed yield per plant (SYP). We conducted a genome-wide epistatic study (GWES), identifying candidate genes that show evidence of SNP-SNP interactions. Although these candidate genes will require further experimental validation, several appear to be involved in developmental processes related to the respective traits. For IN and PH, these include the Dt1 determinacy locus (a soybean meristematic transcription factor), as well as a pectinesterase gene and a squamosa promoter binding gene that in other plants are involved in cell elongation and the vegetative-to-reproductive transition, respectively. For SW, candidate genes include an ortholog of the AP2 gene, which in other species is involved in maintaining seed size, embryo size, seed weight and seed yield. Another SW candidate gene is a histidine phosphotransfer protein - orthologs of which are involved in cytokinin-mediated seed weight regulating pathways. The SYP association loci overlap with regions reported in previous QTL studies to be involved in seed yield.ConclusionsThis study further confirms the utility of GWAS and GWES approaches for identifying marker-trait associations and interactions within a diverse germplasm collection.

Defective Leptotene Chromosome 1 (DLC1) encodes a type-B response regulator and is required for rice meiosis.

Meiosis is critical for sexual reproduction and the generation of new allelic variations in most eukaryotes. In this study, we report the isolation of a meiotic gene, DLC1, using a map-based cloning strategy. The dlc1 mutant is sterile in both male and female gametophytes due to an earlier defect in the leptotene chromosome and subsequent abnormalities at later stages. DLC1 is strongly expressed in the pollen mother cells (PMCs) and tapetum and encodes a nucleus-located rice type-B response regulator (RR) with transcriptional activity. Further investigations showed that DLC1 interacts with all five putative rice histidine phosphotransfer proteins (HPs) in yeast and planta cells, suggesting a possible participation of the two-component signalling systems (TCS) in rice meiosis. Our results demonstrated that DLC1 is required for rice meiosis and fertility, providing useful information for the role of TCS in rice meiosis.

A Genome-Wide Association Study Identifies New Loci Involved in Wound-Induced Lateral Root Formation in Arabidopsis thaliana.

Root systems can display variable architectures that contribute to nutrient foraging or to increase the tolerance of abiotic stress conditions. Root tip excision promotes the developmental progression of previously specified lateral root (LR) founder cells, which allows to easily measuring the branching capacity of a given root as regards its genotype and/or growth conditions. Here, we describe the natural variation among 120 Arabidopsis thaliana accessions in root system architecture (RSA) after root tip excision. Wound-induced changes in RSA were associated with 19 genomic loci using genome-wide association mapping. Three candidate loci associated with wound-induced LR formation were investigated. Sequence variation in the hypothetical protein encoded by the At4g01090 gene affected wound-induced LR development and its loss-of-function mutants displayed a reduced number of LRs after root tip excision. Changes in a histidine phosphotransfer protein putatively involved in cytokinin signaling were significantly associated with LR number variation after root tip excision. Our results provide a better understanding of some of the genetic components involved in LR capacity variation among accessions.

Role of the highly conserved G68 residue in the yeast phosphorelay protein Ypd1: implications for interactions between histidine phosphotransfer (HPt) and response regulator proteins

BackgroundMany bacteria and certain eukaryotes utilize multi-step His-to-Asp phosphorelays for adaptive responses to their extracellular environments. Histidine phosphotransfer (HPt) proteins function as key components of these pathways. HPt proteins are genetically diverse, but share a common tertiary fold with conserved residues near the active site. A surface-exposed glycine at the H + 4 position relative to the phosphorylatable histidine is found in a significant number of annotated HPt protein sequences. Previous reports demonstrated that substitutions at this position result in diminished phosphotransfer activity between HPt proteins and their cognate signaling partners.ResultsWe report the analysis of partner binding interactions and phosphotransfer activity of the prototypical HPt protein Ypd1 from Saccharomyces cerevisiae using a set of H + 4 (G68) substituted proteins. Substitutions at this position with large, hydrophobic, or charged amino acids nearly abolished phospho-acceptance from the receiver domain of its upstream signaling partner, Sln1 (Sln1-R1). An in vitro binding assay indicated that G68 substitutions caused only modest decreases in affinity between Ypd1 and Sln1-R1, and these differences did not appear to be large enough to account for the observed decrease in phosphotransfer activity. The crystal structure of one of these H + 4 mutants, Ypd1-G68Q, which exhibited a diminished ability to participate in phosphotransfer, shows a similar overall structure to that of wild-type. Molecular modelling suggests that the highly conserved active site residues within the receiver domain of Sln1 must undergo rearrangement to accommodate larger H + 4 substitutions in Ypd1.ConclusionsPhosphotransfer reactions require precise arrangement of active site elements to align the donor-acceptor atoms and stabilize the transition state during the reaction. Any changes likely result in an inability to form a viable transition state during phosphotransfer. Our data suggest that the high degree of evolutionary conservation of residues with small side chains at the H + 4 position in HPt proteins is required for optimal activity and that the presence of larger residues at the H + 4 position would cause alterations in the positioning of active site residues in the partner response regulator.

Role of the highly conserved G68 residue in the yeast phosphorelay protein Ypd1: implications for interactions between histidine phosphotransfer (HPt) and response regulator proteins

Role of the highly conserved G68 residue in the yeast phosphorelay protein Ypd1: implications for interactions between histidine phosphotransfer (HPt) and response regulator proteins

The OsRR24/LEPTO1 Type-B Response Regulator is Essential for the Organization of Leptotene Chromosomes in Rice Meiosis.

Response regulators play significant roles in controlling various biological processes; however, their roles in plant meiosis remain unclear. Here, we report the identification of OsRR24/LEPTOTENE1 (LEPTO1), a rice (Oryza sativa) type-B response regulator that participates in the establishment of key molecular and morphological features of chromosomes in leptotene, an early stage of prophase I in meiosis. Although meiosis initiates normally, as indicated by staining of the centromere-specific histone CENH3, the meiotic chromosomes in lepto1 mutant pollen mother cells fail to form the thin thread-like structures that are typical of leptotene chromosomes in wild-type pollen mother cells. Furthermore, lepto1 mutants fail to form chromosomal double-strand breaks, do not recruit meiosis-specific proteins to the meiotic chromosomes, and show disrupted callose deposition. LEPTO1 also is essential for programmed cell death in tapetal cells. LEPTO1 contains a conserved signal receiver domain (DDK) and a myb-like DNA binding domain at the N terminus. LEPTO1 interacts with two authentic histidine phosphotransfer (AHP) proteins, OsAHP1 and OsAHP2, via the DDK domain, and a phosphomimetic mutation of the DDK domain relieves its repression of LEPTO1 transactivation activity. Collectively, our results show that OsRR24/LEPTO1 plays a significant role in the leptotene phase of meiotic prophase I.

The single-domain response regulator LerC functions as a connector protein in the Legionella pneumophila effectors regulatory network.

The intracellular pathogen Legionella pneumophila translocates more than 300 effector proteins into host cells during infection. The PmrAB two-component system (TCS) has been shown to activate the expression of a large pool of these effector-encoding genes (EEGs) and the LetAS TCS, as part of the LetAS-RsmYZ-CsrA cascade, has been shown to repress the expression of another pool of EEGs. We identified a single-domain response regulator (SDRR), named LerC, which functions as a connector protein between the PmrAB and the LetAS TCSs. The lerC gene is strongly activated by the PmrAB TCS and the LerC protein inhibits the activity of the LetAS TCS. The LerC protein specifically interacts with the HPT (histidine-phosphotransfer) domain of LetS, leading to reduced expression of the small RNAs RsmY and RsmZ, which leads to a reduced expression of the pool of EEGs regulated by the LetAS-RsmYZ-CsrA cascade. In addition, the conserved aspartic acid located in the LerC receiver domain is essential for its phosphorylation and function, suggesting that LerC functions as a phosphate-sink of LetS. Our results demonstrate a new role for SDRRs as connector proteins in regulatory networks, suggesting that members of this widespread group of proteins might function as connector proteins in other bacterial regulatory networks.