Histidine Phosphotransfer Proteins Research Articles

A cytokinin response factor PtCRF1 is involved in the regulation of wood formation in poplar.

Wood formation is a complex developmental process under the control of multiple levels of regulatory transcriptional network and hormone signals in trees. It is well known that cytokinin (CK) signaling plays an important role in maintaining the activity of the vascular cambium. The CK response factors (CRFs) encoding a subgroup of AP2 transcription factors have been identified to mediate the CK-dependent regulation in different plant developmental processes. However, the functions of CRFs in wood development remain unclear. Here, we characterized the function of PtCRF1, a CRF transcription factor isolated from poplar, in the process of wood formation. The PtCRF1 is preferentially expressed in secondary vasculature, especially in vascular cambium and secondary phloem, and encodes a transcriptional activator. Overexpression of PtCRF1 in transgenic poplar plants led to a significant reduction in the cell layer number of vascular cambium. The development of wood tissue was largely promoted in the PtCRF1-overexpressing lines, while it was significantly compromised in the CRISPR/Cas9-generated double mutant plants of PtCRF1 and its closest homolog PtCRF2. The RNA sequencing (RNA-seq) and quantitative reverse transcription PCR (RT-qPCR) analyses showed that PtCRF1 repressed the expression of the typical CK-responsive genes. Furthermore, bimolecular fluorescence complementation assays revealed that PtCRF1 competitively inhibits the direct interactions between histidine phosphotransfer proteins and type-B response regulator by binding to PtHP protein. Collectively, these results indicate that PtCRF1 negatively regulates CK signaling and is required for woody cell differentiation in poplar.

Two-Component System Genes in Brassica napus: Identification, Analysis, and Expression Patterns in Response to Abiotic and Biotic Stresses.

The two-component system (TCS), consisting of histidine kinases (HKs), histidine phosphotransfer proteins (HPs) and response regulators (RRs) in eukaryotes, plays pivotal roles in regulating plant growth, development, and responses to environment stimuli. However, the TCS genes were poorly characterized in rapeseed, which is an important tetraploid crop in Brassicaceae. In this work, a total of 182 BnaTCS genes were identified, including 43 HKs, 16 HPs, and 123 RRs, which was more than that in other crops due to segmental duplications during the process of polyploidization. It was significantly different in genetic diversity between the three subfamilies, and some members showed substantial genetic differentiation among the three rapeseed ecotypes. Several hormone- and stress-responsive cis-elements were identified in the putative promoter regions of BnaTCS genes. Furthermore, the expression of BnaTCS genes under abiotic stresses, exogenous phytohormone, and biotic stresses was analyzed, and numerous candidate stress-responsive genes were screened out. Meanwhile, using a natural population with 505 B. napus accessions, we explored the genetic effects of BnaTCS genes on salt tolerance by association mapping analysis and detected some significant association SNPs/genes. The result will help to further understand the functions of TCS genes in the developmental and stress tolerance improvement in B. napus.

Genetic robustness control of auxin output in priming organ initiation

Auxin signaling is essential for organ initiation in plants. How genetic robustness controls auxin output during organ initiation is largely unknown. Here, we identified DORNROSCHEN-LIKE (DRNL) as a target of MONOPTEROS (MP) that plays essential roles in organ initiation. We demonstrate that MP physically interacts with DRNL to inhibit cytokinin accumulation by directly activating ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 6 and CYTOKININ OXIDASE 6. DRN, the paralogous gene of DRNL, acts redundantly with DRNL but is not coexpressed with DRNL in the organ founder cells in which DRNL is expressed. We demonstrate that DRNL directly inhibits DRN expression in the peripheral zone, whereas DRN transcripts are ectopically activated in drnl mutants and fully restore the functional deficiency of drnl in organ initiation. Our results provide a mechanistic framework for the robust control of auxin signaling in organ initiation through paralogous gene-triggered spatial gene compensation effects.

Comparative Omics-Based Identification and Expression Analysis of a Two-Component System in Vigna radiata in Drought Stress

Two-component system (TCS) genes regulate a wide range of biological activities in prokaryotes and eukaryotes, including plants. TCS plays an important role in cellular responses to external stimuli, such as biotic and abiotic factors. In plants, this system supports cell division, leaf senescence, stress response, chloroplast division, and nutrient signaling. There are three kinds of proteins responsible for the appropriate functioning of the TCS system: histidine kinases (HKs), histidine phosphotransfer proteins (HPs), and response regulators (RRs). The results of the current study revealed that Vigna radiata has 54 genes encoding potential TCS proteins, which were divided into three subgroups: 18 HKs, 9 HPs (seven true and two pseudos), and 27 RRs (8 type-A, 8 type-B, 3 type-C, and 8 PRRS). The anticipated TCS genes were widely dispersed across all eleven chromosomes and had family-specific intron/exon structures. After investigating TCS genes in a variety of plant species, we determined that Vigna HK (L)s, HPs, and RRs have closer evolutionary relationships with other legume genes. Gene duplication, including segmental and tandem types, is the most frequent source of gene family expansion. Multiple stress-related cis-elements were predicted in the promoter sequences of the VrTCS genes. RNA-seq data analysis demonstrated that VrTCS genes were expressed in clusters of upregulated and downregulated groups in response to drought stress. Moreover, these clusters were differentially expressed as early or late responses to drought stress. Real-time qPCR showed that VrHK2, VrHK3, VrPHYE, VrHP4.1, VrRR5.2, and VrRR10 genes were upregulated, while VrRR3 and VrHP6.1 genes were downregulated in response to drought stress. The current study highlights the architecture of V. radiata TCS and provides a robust framework for subsequent functional evaluation.

A Survey of Two-Component Systems in Coxiella burnetii Reveals Redundant Regulatory Schemes and a Requirement for an Atypical PhoBR System in Mammalian Cell Infection.

Coxiella burnetii is an obligate intracellular bacterium and the etiological agent of Q fever in humans. C. burnetii transitions between a replicative, metabolically active large-cell variant (LCV) and a spore-like, quiescent small-cell variant (SCV) as a likely mechanism to ensure survival between host cells and mammalian hosts. C. burnetii encodes three canonical two-component systems, four orphan hybrid histidine kinases, five orphan response regulators, and a histidine phosphotransfer protein, which have been speculated to play roles in the signaling required for C. burnetii morphogenesis and virulence. However, very few of these systems have been characterized. By employing a CRISPR interference system for genetic manipulation of C. burnetii, we created single- and multigene transcriptional knockdown strains targeting most of these signaling genes. Through this, we revealed a role for the C. burnetii PhoBR canonical two-component system in virulence, regulation of [Pi] maintenance, and Pi transport. We also outline a novel mechanism by which PhoBR function may be regulated by an atypical PhoU-like protein. We also determined that the GacA.2/GacA.3/GacA.4/GacS orphan response regulators coordinately and disparately regulate expression of SCV-associated genes in C. burnetii LCVs. These foundational results will inform future studies on the role of C. burnetii two-component systems in virulence and morphogenesis. IMPORTANCE C. burnetii is an obligate intracellular bacterium with a spore-like stability allowing it to survive long periods of time in the environment. This stability is likely due to its biphasic developmental cycle, whereby it can transition from an environmentally stable small-cell variant (SCV) to a metabolically active large-cell variant (LCV). Here, we define the role of two-component phosphorelay systems (TCS) in C. burnetii's ability to survive within the harsh environment contained in the phagolysosome of host cells. We show that the canonical PhoBR TCS has an important role in C. burnetii virulence and phosphate sensing. Further examination of the regulons controlled by orphan regulators indicated a role in modulating gene expression of SCV-associated genes, including genes essential for cell wall remodeling.

Genome-wide identification and expression analysis of two-component system genes in sweet potato (Ipomoea batatas L.).

Two-component system (TCS), which comprises histidine kinases (HKs), histidine phosphotransfer proteins (HPs), and response regulators (RRs), plays essential roles in regulating plant growth, development, and response to various environmental stimuli. TCS genes have been comprehensively identified in various plants, while studies on the genome-wide identification and analysis of TCS in sweet potato were still not reported. Therefore, in this study, a total of 90 TCS members consisting of 20 HK(L)s, 11 HPs, and 59 RRs were identified in the genome of Ipomoea batatas. Furthermore, their gene structures, conserved domains, and phylogenetic relationships were analyzed in detail. Additionally, the gene expression profiles in various organs were analyzed, and response patterns to adverse environmental stresses were investigated. The results showed that these 90 TCS genes were mapped on 15 chromosomes with a notably uneven distribution, and the expansion of TCS genes in sweet potato was attributed to both segmental and tandem duplications. The majority of the TCS genes showed distinct organ-specific expression profiles, especially in three types of roots (stem roots, fibrous roots, tuberous roots). Moreover, most of the TCS genes were either induced or suppressed upon treatment with abiotic stresses (drought, salinity, cold, heat) and exogenous phytohormone abscisic acid (ABA). In addition, the yeast-two hybrid system was used to reveal the HK-HP-RR protein-protein interactions. IbHP1, IbHP2, IbHP4, and IbHP5 could interact with three HKs (IbHK1a, IbHK1b, and IbHK5), and also interact with majority of the type-B RRs (IbRR20-IbRR28), while no interaction affinity was detected for IbHP3. Our systematic analyses could provide insights into the characterization of the TCS genes, and further the development of functional studies in sweet potato.

Systematic analysis of Histidine photosphoto transfer gene family in cotton and functional characterization in response to salt and around tolerance

BackgroundPhosphorylation regulated by the two-component system (TCS) is a very important approach signal transduction in most of living organisms. Histidine phosphotransfer (HP) is one of the important members of the TCS system. Members of the HP gene family have implications in plant stresses tolerance and have been deeply studied in several crops. However, upland cotton is still lacking with complete systematic examination of the HP gene family.ResultsA total of 103 HP gene family members were identified. Multiple sequence alignment and phylogeny of HPs distributed them into 7 clades that contain the highly conserved amino acid residue “XHQXKGSSXS”, similar to the Arabidopsis HP protein. Gene duplication relationship showed the expansion of HP gene family being subjected with whole-genome duplication (WGD) in cotton. Varying expression profiles of HPs illustrates their multiple roles under altering environments particularly the abiotic stresses. Analysis is of transcriptome data signifies the important roles played by HP genes against abiotic stresses. Moreover, protein regulatory network analysis and VIGS mediated functional approaches of two HP genes (GhHP23 and GhHP27) supports their predictor roles in salt and drought stress tolerance.ConclusionsThis study provides new bases for systematic examination of HP genes in upland cotton, which formulated the genetic makeup for their future survey and examination of their potential use in cotton production.

The histidine phosphotransfer AHP4 plays a negative role in Arabidopsis plant response to drought

Cytokinin plays an important role in plant stress responses via a multistep signaling pathway, involving the histidine phosphotransfer proteins (HPs). In Arabidopsis thaliana, the AHP2, AHP3 and AHP5 proteins are known to affect drought responses; however, the role of AHP4 in drought adaptation remains undetermined. In the present study, using a loss-of-function approach we showed that AHP4 possesses an important role in the response of Arabidopsis to drought. This is evidenced by the higher survival rates of ahp4 than wild-type (WT) plants under drought conditions, which is accompanied by the downregulated AHP4 expression in WT during periods of dehydration. Comparative transcriptome analysis of ahp4 and WT plants revealed AHP4-mediated expression of several dehydration- and/or abscisic acid-responsive genes involved in modulation of various physiological and biochemical processes important for plant drought acclimation. In comparison with WT, ahp4 plants showed increased wax crystal accumulation in stems, thicker cuticles in leaves, greater sensitivity to exogenous abscisic acid at germination, narrow stomatal apertures, heightened leaf temperatures during dehydration, and longer root length under osmotic stress. In addition, ahp4 plants showed greater photosynthetic efficiency, lower levels of reactive oxygen species, reduced electrolyte leakage and lipid peroxidation, and increased anthocyanin contents under drought, when compared with WT. These differences displayed in ahp4 plants are likely due to upregulation of genes that encode enzymes involved in reactive oxygen species scavenging and non-enzymatic antioxidant metabolism. Overall, our findings suggest that AHP4 plays a crucial role in plant drought adaptation.

Transcriptome Analysis Reveals Genetic Factors Related to Callus Induction in Barley

Barley is an important cereal crop worldwide. Its genetic transformation is now limited to very few cultivars because of the high genotype dependence of embryogenic callus. To reveal the key genes or factors controlling the callus induction and plantlet regeneration in barley, we compared the transcriptomic profiles of immature embryos of Golden Promise and ZU9, which differed dramatically in the efficiency of the genetic transformation. The samples were taken at 0, 5, 10 and 20 days of the culture, respectively. In total, 5386 up-regulated and 6257 down-regulated genes were identified in Golden Promise. Several genes, identified exclusively in GP callus, were selected for further investigation. These genes were mainly involved in protein metabolism, energy metabolism, stress response, detoxification and ubiquitin–proteasome. Four YUCCA flavin monooxygenases, one PIN-FORMED, one tryptophan aminotransferase related, three small auxin up RNA, three indole-3-acetic acid and one adenylate isopentenyl transferase, seven cytokinin oxidase/dehydrogenase, three Arabidopsis histidine kinase, three Arabidopsis histidine phosphotransfer protein, and one Arabidopsis response regulator were differentially expressed in the calli of the two barley genotypes, suggesting that biosynthesis, response and transport of auxin and cytokinin might be associated with cell reprogramming during callus induction. The current results provide insights into molecular mechanisms of callus induction at an early developmental stage and are helpful for optimizing the tissue culture system in barley.

Transcriptome analysis of Magnaporthe oryzae reveals the role of histidine phosphotransferase in pathogenesis and stress adaptation

Transcriptome analysis of Magnaporthe oryzae reveals the role of histidine phosphotransferase in pathogenesis and stress adaptation

Two-Component System Genes in Sorghum bicolor: Genome-Wide Identification and Expression Profiling in Response to Environmental Stresses.

The two-component signal transduction system (TCS) acts in a variety of physiological processes in lower organisms and has emerged as a key signaling system in both prokaryotes and eukaryotes, including plants. TCS genes assist plants in processes such as stress resistance, cell division, nutrition signaling, leaf senescence, and chloroplast division. In plants, this system is composed of three types of proteins: response regulators (RRs), histidine kinases (HKs), and histidine phosphotransfer proteins (HPs). We aimed to study the Sorghum bicolor genome and identified 37 SbTCS genes consisting of 13 HKs, 5 HPs, and 19 RRs (3 type-A RRs, 7 type-B RRs, 2 type-C RRs, and 7 pseudo-RRs). The structural and phylogenetic comparison of the SbTCS members with their counterparts in Arabidopsis thaliana, Oryza sativa, Cicer arietinum, and Glycine max showed group-specific conservations and variations. Expansion of the gene family members is mostly a result of gene duplication, of both the tandem and segmental types. HKs and RRs were observed to be originated from segmental duplication, while some HPs originated from tandem duplication. The nuclear genome of S. bicolor contain 10 chromosomes and these SbTCS genes are randomly distributed on all the chromosomes. The promoter sequences of the SbTCS genes contain several abiotic stress-related cis-elements. RNA-seq and qRT-PCR-based expression analysis demonstrated most of the TCS genes were responsive to drought and salt stresses in leaves, which suggest their role in leaf development. This study lays a foundation for further functional study of TCS genes for stress tolerance and developmental improvement in S. bicolor.

Evaluation of type-B RR dimerization in poplar: A mechanism to preserve signaling specificity?

Plants possess specific signaling pathways, such as the MultiStep Phosphorelay (MSP), which is involved in cytokinin and ethylene sensing, and light, drought or osmotic stress sensing. These MSP comprise histidine-aspartate kinases (HKs) as receptors, histidine phosphotransfer (HPts) proteins acting as phosphorelay proteins, and response regulators (RRs), some of which act as transcription factors (type-B RRs). In previous studies, we identified partners of the poplar osmosensing signaling pathway, composed of two HKs, three main HPts, and six type-B RRs. To date, it is unresolved as to how cytokinin or osmotic stress signal specificity is achieved in the MSP in order to generate specific responses. Here, we present a large-scale interaction study of poplar type-B RR dimerization. Using the two-hybrid assay, we were able to show the homodimerization of type-B RRs, the heterodimerization of duplicated type-B RRs, and surprisingly, a lack of interaction between some type-B RRs belonging to different duplicates. The lack of interaction of the duplicates RR12-14 and RR18-19, which are involved in the osmosensing pathway has been confirmed by BiFC experiments. This study reveals, for the first time, an overview of type-B RR dimerization in poplar and makes way for the hypothesis that signal specificity for cytokinin or osmotic stress could be in part due to the fact that it is impossible for specific type-B RRs to heterodimerize.

A cryptic inhibitor of cytokinin phosphorelay controls rice grain size

Plant hormone cytokinin signals through histidine-aspartic acid (H-D) phosphorelay to regulate plant growth and development. While it is well known that the phosphorelay involves histidine kinases, histidine phosphotransfer proteins (HPs), and response regulators (RRs), how this process is regulated by external components remains unknown. Here we demonstrate that protein phosphatase with kelch-like domains (PPKL1), known as a signaling component of steroid hormone brassinosteroid, is actually a cryptic inhibitor of cytokinin phosphorelay in rice (Oryza sativa). Mutation at a specific amino acid D364 of PPKL1 activates cytokinin response and thus enlarges grain size in a semi-dominant mutant named s48. Overexpression of PPKL1 containing D364, either with the deletion of the phosphatase domain or not, rescues the s48 mutant phenotype. PPKL1 interacts with OsAHP2, one of authentic HPs, and D364 resides in a region resembling the receiver domain of RRs. Accordingly, PPKL1 can utilize D364 to suppress OsAHP2-to-RR phosphorelay, whereas mutation of D364 abolishes the effect. This function of PPKL1 is independent of the phosphatase domain that is required for brassinosteroid signaling. Importantly, editing of the D364-residential region produces a diversity of semi-dominant mutations associated with variously increased grain sizes. Further screening of the edited plants enables the identification of two genotypes that confer significantly improved grain yield. Collectively, our study uncovers a noncanonical cytokinin signaling suppressor and provides a robust tool for seed rational design.

RetS inhibits Pseudomonas aeruginosa biofilm formation by disrupting the canonical histidine kinase dimerization interface of GacS

Bacterial signaling histidine kinases (HKs) have long been postulated to function exclusively through linear signal transduction chains. However, several HKs have recently been shown to form complex multikinase networks (MKNs). The most prominent MKN, involving the enzymes RetS and GacS, controls the switch between the motile and biofilm lifestyles in the pathogenic bacterium Pseudomonas aeruginosa. While GacS promotes biofilm formation, RetS counteracts GacS using three distinct mechanisms. Two are dephosphorylating mechanisms. The third, a direct binding between the RetS and GacS HK regions, blocks GacS autophosphorylation. Focusing on the third mechanism, we determined the crystal structure of a cocomplex between the HK region of RetS and the dimerization and histidine phosphotransfer (DHp) domain of GacS. This is the first reported structure of a complex between two distinct bacterial signaling HKs. In the complex, the canonical HK homodimerization interface is replaced by a strikingly similar heterodimeric interface between RetS and GacS. We further demonstrate that GacS autophosphorylates in trans, thus explaining why the formation of a RetS-GacS complex inhibits GacS autophosphorylation. Using mutational analysis in conjunction with bacterial two-hybrid and biofilm assays, we not only corroborate the biological role of the observed RetS-GacS interactions, but also identify a residue critical for the equilibrium between the RetS-GacS complex and the respective RetS and GacS homodimers. Collectively, our findings suggest that RetS and GacS form a domain-swapped hetero-oligomer during the planktonic growth phase of P. aeruginosa before unknown signals cause its dissociation and a relief of GacS inhibition to promote biofilm formation.

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.

High throughput identification of miRNAs reveal novel interacting targets regulating chickpea-rhizobia symbiosis

Legumes developed symbiotic associations to meet its nitrogen requirement. The nitrogen fixation takes place in root nodules which involves bacterial colonization, organogenesis and nitrogen fixation. In order to unravel the miRNA mediated regulation of chickpea symbiosis, one microRNA and four parallel analysis of RNA ends (PARE) libraries were sequenced. Analysis of microRNA library identified a set of 91 miRNAs comprising of 84 conserved and 7 novel miRNAs. Additionally, PARE library analysis revealed 564 genes being targeted by 85 miRNAs. Phylogenetic analysis of the precursor sequences of the 91 miRNAs was carried out which revealed their ancestral relationships. Further, the mechanism of miRNAs biogenesis was predicted using the miRNAs information from other legumes. Reads from the nodule library were mapped to bacterial genomes to predict bacterial-encoded small RNAs. Real time expression analysis was used to validate the antagonistic expression pattern of important miRNA-mRNA target pairs. Four candidate miRNAs were selected for in planta study based on the antagonistic expression profiling as well as the novelty of their respective targets. miR171f, miR172c, miR394 and miR1509 targeted nodulation receptor kinase, Apetala2, histidine phosphotransferase, adenylate kinase respectively and were ectopically expressed in chickpea roots. The overexpression lines showed significant change in nodule numbers, the miR172c, miR394 and miR1509 resulted in an increase in nodule number whereas, miR171f overexpression led to a decrease in nodule number. Our analysis lays the foundation for functional characterization of novel miRNAs and their respective target pairs which control nodulation in chickpea and other leguminous crops.

Two-Component Signaling Systems Regulate Diverse Virulence-Associated Traits in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that can cause problematic infections at different sites throughout the human body. P. aeruginosa encodes a large suite of over 60 two-component signaling systems that enable cells to rapidly sense and respond to external signals. Previous work has shown that some of these sensory systems contribute to P. aeruginosa pathogenesis, but the virulence-associated processes and phenotypic traits that each of these systems controls are still largely unclear. To aid investigations of these sensory systems, we have generated deletion strains for each of 64 genes encoding histidine kinases and one histidine phosphotransferase in P. aeruginosa PA14. We carried out initial phenotypic characterizations of this collection by assaying these mutants for over a dozen virulence-associated traits, and we found that each of these phenotypes is regulated by multiple sensory systems. Our work highlights the usefulness of this collection for further studies of P. aeruginosa two-component signaling systems and provides insight into how these systems may contribute to P. aeruginosa infection.IMPORTANCEPseudomonas aeruginosa can grow and survive under a wide range of conditions, including as a human pathogen. As such, P. aeruginosa must be able to sense and respond to diverse signals and cues in its environment. This sensory capability is endowed in part by the hundreds of two-component signaling proteins encoded in the P. aeruginosa genome, but the precise roles of each remain poorly defined. To facilitate systematic study of the signaling repertoire of P. aeruginosa PA14, we generated a library of deletion strains, each lacking one of the 64 histidine kinases. By subjecting these strains to a battery of phenotypic assays, we confirmed the functions of many and unveiled roles for dozens of previously uncharacterized histidine kinases in controlling various traits, many of which are associated with P. aeruginosa virulence. Thus, this work provides new insight into the functions of two-component signaling proteins and provides a resource for future investigations.

ITRAQ-based comparative proteome analyses of different growth stages revealing the regulatory role of reactive oxygen species in the fruiting body development of Ophiocordyceps sinensis.

In this study, using an isobaric tags for relative and absolute quantitation (iTRAQ ) approach coupled with LC-MS / MS and bioinformatics, the proteomes were analyzed for the crucial three stages covering the fruiting body development of Ophiocordyceps sinensis, including sclerotium (ST), primordium (PR) and mature fruiting body (MF), with a focus on fruiting body development-related proteins and the potential mechanisms of the development. A total of 1,875 proteins were identified. Principal Component Analysis (PCA) demonstrated that the protein patterns between PR and MF were more similar than ST. Differentially accumulated proteins (DAPs) analysis showed that there were 510, 173 and 514 DAPs in the comparisons of ST vs. PR, PR vs. MF and ST vs. MF, respectively. A total of 62 shared DAPs were identified and primarily enriched in proteins related to ‘carbon transport and mechanism’, ‘the response to oxidative stress’, ‘antioxidative activity’ and ‘translation’. KEGG and GO databases showed that the DAPs were enriched in terms of ‘primary metabolisms (amino acid/fatty acid/energy metabolism)’, ‘the response to oxidative stress’ and ‘peroxidase’. Furthermore, 34 DAPs involved in reactive oxygen species (ROS) metabolism were identified and clustered across the three stages using hierarchical clustering implemented in hCluster R package . It was suggested that their roles and the underlying mechanisms may be stage-specific. ROS may play a role in fungal pathogenicity in ST, the fruit-body initiation in PR, sexual reproduction and highland adaptation in MF. Crucial ROS-related proteins were identified, such as superoxide dismutase (SOD, T5A6F1), Nor-1 (T5AFX3), electron transport protein (T5AHD1), histidine phosphotransferase (HPt, T5A9Z5) and Glutathione peroxidase (T5A9V1). Besides, the accumulation of ROS at the three stages were assayed using 2,7-dichlorofuorescin diacetate (DCFH-DA) stanning. A much stronger ROS accumulation was detected at the stage MF, compared to the stages of PR and ST. Sections of ST and fruit-body part of MF were stained by DCFH-DA and observed under the fluorescencemicroscope, showing ROS was distributed within the conidiospore and ascus. Besides, SOD activity increased across the three stages, while CAT activity has a strong increasement in MF compared to the stages of ST and PR. It was suggested that ROS may act in gradient-dependent manner to regulate the fruiting body development. The coding region sequences of six DAPs were analyzed at mRNA level by quantitative real-time PCR (qRT-PCR). The results support the result of DAPs analysis and the proteome sequencing data. Our findings offer the perspective of proteome to understand the biology of fruiting body development and highland adaptation in O. sinensis, which would inform the big industry of this valuable fungus.

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.