Insights into Phakopsora pachyrhizi Effector-Effector Interactions.

The multifaceted role of pathogen-encoded effectors in plant-pathogen interactions is complex and not fully understood. Effectors operate within intricate host environments, interacting with host proteins and other effectors to modulate virulence. The complex interplay between effectors raises the concept of metaeffectors, wherein some effectors regulate the activity of others. While previous research has demonstrated the importance of effector repertoires in pathogen virulence, only a limited number of studies have investigated the interactions between these effectors. This study explores the interactions among Phakopsora pachyrhizi effector candidates (PpECs). P. pachyrhizi haustorial transcriptome analysis identified a collection of predicted PpECs. Among these, PpEC23 was found to interact with PpEC48, prompting further exploration into their potential interaction with other effectors. Here, we utilized a yeast two-hybrid screen to explore protein-protein interactions between PpECs. A split-luciferase complementation assay also demonstrated that these interactions could occur within soybean cells. Interestingly, PpEC48 displayed the ability to interact with several small cysteine-rich proteins (SCRPs), suggesting its affinity for this specific class of effectors. We show that these interactions involve a histidine-rich domain within PpEC48, emphasizing the significance of structural motifs in mediating effector interactions. The unique nature of PpEC48, showing no sequence matches in other organisms, suggests its relatively recent evolution and potential orphan gene status. Our work reveals insights into the intricate network of interactions among P. pachyrhizi effector-effector interactions. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Increased signal-to-noise ratios within experimental field trials by regressing spatially distributed soil properties as principal components.

Environmental variability poses a major challenge to any field study. Researchers attempt to mitigate this challenge through replication. Thus, the ability to detect experimental signals is determined by the degree of replication and the amount of environmental variation, noise, within the experimental system. A major source of noise in field studies comes from the natural heterogeneity of soil properties which create microtreatments throughout the field. In addition, the variation within different soil properties is often nonrandomly distributed across a field. We explore this challenge through a sorghum field trial dataset with accompanying plant, microbiome, and soil property data. Diverse sorghum genotypes and two watering regimes were applied in a split-plot design. We describe a process of identifying, estimating, and controlling for the effects of spatially distributed soil properties on plant traits and microbial communities using minimal degrees of freedom. Importantly, this process provides a method with which sources of environmental variation in field data can be identified and adjusted, improving our ability to resolve effects of interest and to quantify subtle phenotypes.

Identification of beneficial and detrimental bacteria impacting sorghum responses to drought using multi-scale and multi-system microbiome comparisons.

Drought is a major abiotic stress limiting agricultural productivity. Previous field-level experiments have demonstrated that drought decreases microbiome diversity in the root and rhizosphere. How these changes ultimately affect plant health remains elusive. Toward this end, we combined reductionist, transitional and ecological approaches, applied to the staple cereal crop sorghum to identify key root-associated microbes that robustly affect drought-stressed plant phenotypes. Fifty-three Arabidopsis-associated bacteria were applied to sorghum seeds and their effect on root growth was monitored. Two Arthrobacter strains caused root growth inhibition (RGI) in Arabidopsis and sorghum. In the context of synthetic communities, Variovorax strains were able to protect plants from Arthrobacter-caused RGI. As a transitional system, high-throughput phenotyping was used to test the synthetic communities. During drought stress, plants colonized by Arthrobacter had reduced growth and leaf water content. Plants colonized by both Arthrobacter and Variovorax performed as well or better than control plants. In parallel, we performed a field trial wherein sorghum was evaluated across drought conditions. By incorporating data on soil properties into the microbiome analysis, we accounted for experimental noise with a novel method and were able to observe the negative correlation between the abundance of Arthrobacter and plant growth. Having validated this approach, we cross-referenced datasets from the high-throughput phenotyping and field experiments and report a list of bacteria with high confidence that positively associated with plant growth under drought stress. In conclusion, a three-tiered experimental system successfully spanned the lab-to-field gap and identified beneficial and deleterious bacterial strains for sorghum under drought.

GmNF-YC4-2 Increases Protein, Exhibits Broad Disease Resistance and Expedites Maturity in Soybean.

The NF-Y gene family is a highly conserved set of transcription factors. The functional transcription factor complex is made up of a trimer between NF-YA, NF-YB, and NF-YC proteins. While mammals typically have one gene for each subunit, plants often have multigene families for each subunit which contributes to a wide variety of combinations and functions. Soybean plants with an overexpression of a particular NF-YC isoform GmNF-YC4-2 (Glyma.04g196200) in soybean cultivar Williams 82, had a lower amount of starch in its leaves, a higher amount of protein in its seeds, and increased broad disease resistance for bacterial, viral, and fungal infections in the field, similar to the effects of overexpression of its isoform GmNF-YC4-1 (Glyma.06g169600). Interestingly, GmNF-YC4-2-OE (overexpression) plants also filled pods and senesced earlier, a novel trait not found in GmNF-YC4-1-OE plants. No yield difference was observed in GmNF-YC4-2-OE compared with the wild-type control. Sequence alignment of GmNF-YC4-2, GmNF-YC4-1 and AtNF-YC1 indicated that faster maturation may be a result of minor sequence differences in the terminal ends of the protein compared to the closely related isoforms.

Candidate Effectors From Uromyces appendiculatus, the Causal Agent of Rust on Common Bean, Can Be Discriminated Based on Suppression of Immune Responses.

Rust fungi are devastating pathogens for several important crop plants. The biotrophic lifestyle of rust fungi requires that they influence their host plants to create a favorable environment for growth and reproduction. Rust fungi secrete a variety of effector proteins that manipulate host target proteins to alter plant metabolism and suppress defense responses. Because of the obligate biotrophic lifestyle of rust fungi, direct evidence for effector function is difficult to obtain, and so suites of experiments utilizing expression in heterologous systems are necessary. Here, we present results from a yeast cell death suppression assay and assays for suppression of PAMP-triggered immunity (PTI) and effector triggered immunity (ETI) based on delivery of effectors through the bacterial type III secretion system. In addition, subcellular localization was tested using transient expression of GFP fusion proteins in Nicotiana benthamiana through Agrobacterium infiltration. We tested 31 representative effector candidates from the devastating common bean rust pathogen Uromyces appendiculatus. These effector candidates were selected based on features of their gene families, most important lineage specificity. We show that several of our effector candidates suppress plant defense. Some of them also belong to families of effector candidates that are present in multiple rust species where their homologs probably also have effector functions. In our analysis of candidate effector mRNA expression, some of those effector candidates that gave positive results in the other assays were not up-regulated during plant infection, indicating that either these proteins have functions at multiple life stages or that strong up-regulation of RNA level in planta may not be as important a criterion for identifying effectors as previously thought. Overall, our pipeline for selecting effector candidates based on sequence features followed by screening assays using heterologous expression systems was successful in discriminating effector candidates. This work lays the foundation for functional characterization of U. appendiculatus effectors, the identification of effector targets, and identification of novel sources for resistance in common bean.

Engineering a Decoy Substrate in Soybean to Enable Recognition of the Soybean Mosaic Virus NIa Protease.

In Arabidopsis, recognition of the AvrPphB effector protease from Pseudomonas syringae is mediated by the disease resistance (R) protein RPS5, which is activated by AvrPphB-induced cleavage of the Arabidopsis protein kinase PBS1. The recognition specificity of RPS5 can be altered by substituting the AvrPphB cleavage site within PBS1 with cleavage sequences for other proteases, including proteases from viruses. AvrPphB also activates defense responses in soybean (Glycine max), suggesting that soybean may contain an R protein analogous to RPS5. It was unknown, however, whether this response is mediated by cleavage of a soybean PBS1-like protein. Here, we show that soybean contains three PBS1 orthologs and that their products are cleaved by AvrPphB. Further, transient expression of soybean PBS1 derivatives containing a five-alanine insertion at their AvrPphB cleavage sites activated cell death in soybean protoplasts, demonstrating that soybean likely contains an AvrPphB-specific resistance protein that is activated by a conformational change in soybean PBS1 proteins. Significantly, we show that a soybean PBS1 decoy protein modified to contain a cleavage site for the soybean mosaic virus (SMV) NIa protease triggers cell death in soybean protoplasts when cleaved by this protease, indicating that the PBS1 decoy approach will work in soybean, using endogenous PBS1 genes. Lastly, we show that activation of the AvrPphB-dependent cell death response effectively inhibits systemic spread of SMV in soybean. These data also indicate that decoy engineering may be feasible in other crop plant species that recognize AvrPphB protease activity.

QQS orphan gene and its interactor NF-YC4 reduce susceptibility to pathogens and pests.

Enhancing the nutritional quality and disease resistance of crops without sacrificing productivity is a key issue for developing varieties that are valuable to farmers and for simultaneously improving food security and sustainability. Expression of the Arabidopsis thaliana species-specific AtQQS (Qua-Quine Starch) orphan gene or its interactor, NF-YC4 (Nuclear Factor Y, subunit C4), has been shown to increase levels of leaf/seed protein without affecting the growth and yield of agronomic species. Here, we demonstrate that overexpression of AtQQS and NF-YC4 in Arabidopsis and soybean enhances resistance/reduces susceptibility to viruses, bacteria, fungi, aphids and soybean cyst nematodes. A series of Arabidopsis mutants in starch metabolism were used to explore the relationships between QQS expression, carbon and nitrogen partitioning, and defense. The enhanced basal defenses mediated by QQS were independent of changes in protein/carbohydrate composition of the plants. We demonstrate that either AtQQS or NF-YC4 overexpression in Arabidopsis and in soybean reduces susceptibility of these plants to pathogens/pests. Transgenic soybean lines overexpressing NF-YC4 produce seeds with increased protein while maintaining healthy growth. Pull-down studies reveal that QQS interacts with human NF-YC, as well as with Arabidopsis NF-YC4, and indicate two QQS binding sites near the NF-YC-histone-binding domain. A new model for QQS interaction with NF-YC is speculated. Our findings illustrate the potential of QQS and NF-YC4 to increase protein and improve defensive traits in crops, overcoming the normal growth-defense trade-offs.

Suppression or Activation of Immune Responses by Predicted Secreted Proteins of the Soybean Rust Pathogen Phakopsora pachyrhizi.

Rust fungi, such as the soybean rust pathogen Phakopsora pachyrhizi, are major threats to crop production. They form specialized haustoria that are hyphal structures intimately associated with host-plant cell membranes. These haustoria have roles in acquiring nutrients and secreting effector proteins that manipulate host immune systems. Functional characterization of effector proteins of rust fungi is important for understanding mechanisms that underlie their virulence and pathogenicity. Hundreds of candidate effector proteins have been predicted for rust pathogens, but it is not clear how to prioritize these effector candidates for further characterization. There is a need for high-throughput approaches for screening effector candidates to obtain experimental evidence for effector-like functions, such as the manipulation of host immune systems. We have focused on identifying effector candidates with immune-related functions in the soybean rust fungus P. pachyrhizi. To facilitate the screening of many P. pachyrhizi effector candidates (named PpECs), we used heterologous expression systems, including the bacterial type III secretion system, Agrobacterium infiltration, a plant virus, and a yeast strain, to establish an experimental pipeline for identifying PpECs with immune-related functions and establishing their subcellular localizations. Several PpECs were identified that could suppress or activate immune responses in nonhost Nicotiana benthamiana, N. tabacum, Arabidopsis, tomato, or pepper plants.

Prediction of the in planta Phakopsora pachyrhizi secretome and potential effector families.

Asian soybean rust (ASR), caused by the obligate biotrophic fungus Phakopsora pachyrhizi, can cause losses greater than 80%. Despite its economic importance, there is no soybean cultivar with durable ASR resistance. In addition, the P. pachyrhizi genome is not yet available. However, the availability of other rust genomes, as well as the development of sample enrichment strategies and bioinformatics tools, has improved our knowledge of the ASR secretome and its potential effectors. In this context, we used a combination of laser capture microdissection (LCM), RNAseq and a bioinformatics pipeline to identify a total of 36 350 P. pachyrhizi contigs expressed in planta and a predicted secretome of 851 proteins. Some of the predicted secreted proteins had characteristics of candidate effectors: small size, cysteine rich, do not contain PFAM domains (except those associated with pathogenicity) and strongly expressed in planta. A comparative analysis of the predicted secreted proteins present in Pucciniales species identified new members of soybean rust and new Pucciniales- or P. pachyrhizi-specific families (tribes). Members of some families were strongly up-regulated during early infection, starting with initial infection through haustorium formation. Effector candidates selected from two of these families were able to suppress immunity in transient assays, and were localized in the plant cytoplasm and nuclei. These experiments support our bioinformatics predictions and show that these families contain members that have functions consistent with P. pachyrhizi effectors.

A Small Cysteine-Rich Protein from the Asian Soybean Rust Fungus, Phakopsora pachyrhizi, Suppresses Plant Immunity.

The Asian soybean rust fungus, Phakopsora pachyrhizi, is an obligate biotrophic pathogen causing severe soybean disease epidemics. Molecular mechanisms by which P. pachyrhizi and other rust fungi interact with their host plants are poorly understood. The genomes of all rust fungi encode many small, secreted cysteine-rich proteins (SSCRP). While these proteins are thought to function within the host, their roles are completely unknown. Here, we present the characterization of P. pachyrhizi effector candidate 23 (PpEC23), a SSCRP that we show to suppress plant immunity. Furthermore, we show that PpEC23 interacts with soybean transcription factor GmSPL12l and that soybean plants in which GmSPL12l is silenced have constitutively active immunity, thereby identifying GmSPL12l as a negative regulator of soybean defenses. Collectively, our data present evidence for a virulence function of a rust SSCRP and suggest that PpEC23 is able to suppress soybean immune responses and physically interact with soybean transcription factor GmSPL12l, a negative immune regulator.

Molecular Soybean-Pathogen Interactions.

Soybean hosts a wide variety of pathogens that cause significant yield losses. The importance of soybean as a major oilseed crop has led to research focused on its interactions with pathogens, such as Soybean mosaic virus, Pseudomonas syringae, Phytophthora sojae, Phakopsora pachyrhizi, and Heterodera glycines. Pioneering work on soybean's interactions with these organisms, which represent the five major pathogen groups (viruses, bacteria, oomycetes, fungi, and nematodes), has contributed to our understanding of the molecular mechanisms underlying virulence and immunity. These mechanisms involve conserved and unique features that validate the need for research in both soybean and homologous model systems. In this review, we discuss identification of effectors and their functions as well as resistance gene-mediated recognition and signaling. We also point out areas in which model systems and recent advances in resources and tools have provided opportunities to gain deeper insights into soybean-pathogen interactions.

Arabidopsis miR827 mediates post-transcriptional gene silencing of its ubiquitin E3 ligase target gene in the syncytium of the cyst nematode Heterodera schachtii to enhance susceptibility.

MicroRNAs (miRNAs) are a major class of small non-coding RNAs with emerging functions in biotic and abiotic interactions. Here, we report on a new functional role of Arabidopsis miR827 and its NITROGEN LIMITATION ADAPTATION (NLA) target gene in mediating plant susceptibility to the beet cyst nematode Heterodera schachtii. Cyst nematodes are sedentary endoparasites that induce the formation of multinucleated feeding structures termed syncytia in the roots of host plants. Using promoter:GUS fusion assays we established that miR827 was activated in the initial feeding cells and this activation was maintained in the syncytium during all sedentary stages of nematode development. Meanwhile, the NLA target gene, which encodes an ubiquitin E3 ligase enzyme, was post-transcriptionally silenced in the syncytium to permanently suppress its activity during all nematode parasitic stages. Overexpression of miR827 in Arabidopsis resulted in hyper-susceptibility to H. schachtii. In contrast, inactivation of miR827 activity through target mimicry or by overexpression a miR827-resistant cDNA of NLA produced the opposite phenotype of reduced plant susceptibility to H. schachtii. Gene expression analysis of several pathogenesis-related genes together with Agrobacterium-mediated transient expression in Nicotiana benthamiana provided strong evidence that miR827-mediated downregulation of NLA to suppress basal defense pathways. In addition, using yeast two-hybrid screens we identified several candidates of NLA-interacting proteins that are involved in a wide range of biological processes and molecular functions, including three pathogenesis-related proteins. Taken together, we conclude that nematode-activated miR827 in the syncytium is necessary to suppress immune responses in order to establish infection and cause disease.

A Plasmodium-like virulence effector of the soybean cyst nematode suppresses plant innate immunity.

Heterodera glycines, the soybean cyst nematode, delivers effector proteins into soybean roots to initiate and maintain an obligate parasitic relationship. HgGLAND18 encodes a candidate H. glycines effector and is expressed throughout the infection process. We used a combination of molecular, genetic, bioinformatic and phylogenetic analyses to determine the role of HgGLAND18 during H. glycines infection. HgGLAND18 is necessary for pathogenicity in compatible interactions with soybean. The encoded effector strongly suppresses both basal and hypersensitive cell death innate immune responses, and immunosuppression requires the presence and coordination between multiple protein domains. The N-terminal domain in HgGLAND18 contains unique sequence similarity to domains of an immunosuppressive effector of Plasmodium spp., the malaria parasites. The Plasmodium effector domains functionally complement the loss of the N-terminal domain from HgGLAND18. In-depth sequence searches and phylogenetic analyses demonstrate convergent evolution between effectors from divergent parasites of plants and animals as the cause of sequence and functional similarity.

Arabidopsis and Brachypodium distachyon transgenic plants expressing Aspergillus nidulans acetylesterases have decreased degree of polysaccharide acetylation and increased resistance to pathogens.

The plant cell wall has many significant structural and physiological roles, but the contributions of the various components to these roles remain unclear. Modification of cell wall properties can affect key agronomic traits such as disease resistance and plant growth. The plant cell wall is composed of diverse polysaccharides often decorated with methyl, acetyl, and feruloyl groups linked to the sugar subunits. In this study, we examined the effect of perturbing cell wall acetylation by making transgenic Arabidopsis (Arabidopsis thaliana) and Brachypodium (Brachypodium distachyon) plants expressing hemicellulose- and pectin-specific fungal acetylesterases. All transgenic plants carried highly expressed active Aspergillus nidulans acetylesterases localized to the apoplast and had significant reduction of cell wall acetylation compared with wild-type plants. Partial deacetylation of polysaccharides caused compensatory up-regulation of three known acetyltransferases and increased polysaccharide accessibility to glycosyl hydrolases. Transgenic plants showed increased resistance to the fungal pathogens Botrytis cinerea and Bipolaris sorokiniana but not to the bacterial pathogens Pseudomonas syringae and Xanthomonas oryzae. These results demonstrate a role, in both monocot and dicot plants, of hemicellulose and pectin acetylation in plant defense against fungal pathogens.

Genome-wide identification of genes regulated by the Rcs phosphorelay system in Erwinia amylovora.

The exopolysaccharide amylovoran is one of the major pathogenicity factors in Erwinia amylovora, the causal agent of fire blight of apples and pears. We have previously demonstrated that the RcsBCD phosphorelay system is essential for virulence by controlling amylovoran biosynthesis. We have also found that the hybrid sensor kinase RcsC differentially regulates amylovoran production in vitro and in vivo. To further understand how the Rcs system regulates E. amylovora virulence gene expression, we conducted genome-wide microarray analyses to determine the regulons of RcsB and RcsC in liquid medium and on immature pear fruit. Array analyses identified a total of 648 genes differentially regulated by RcsCB in vitro and in vivo. Consistent with our previous findings, RcsB acts as a positive regulator in both conditions, while RcsC positively controls expression of amylovoran biosynthetic genes in vivo but negatively controls expression in vitro. Besides amylovoran biosynthesis and regulatory genes, cell-wall and cell-envelope (membrane) as well as regulatory genes were identified as the major components of the RcsBC regulon, including many novel genes. We have also demonstrated that transcripts of rcsA, rcsC, and rcsD genes but not the rcsB gene were up-regulated when bacterial cells were grown in minimal medium or following infection of pear fruits compared with those grown in Luria Bertani medium. Furthermore, using the genome of E. amylovora ATCC 49946, a hidden Markov model predicted 60 genes with a candidate RcsB binding site in the intergenic region, 28 of which were identified in the microarray assay. Based on these findings as well as previous reported data, a working model has been proposed to illustrate how the Rcs phosphorelay system regulates virulence gene expression in E. amylovora.

Genome sequence analyses of Pseudomonas savastanoi pv. glycinea and subtractive hybridization-based comparative genomics with nine pseudomonads.

Bacterial blight, caused by Pseudomonas savastanoi pv. glycinea (Psg), is a common disease of soybean. In an effort to compare a current field isolate with one isolated in the early 1960s, the genomes of two Psg strains, race 4 and B076, were sequenced using 454 pyrosequencing. The genomes of both Psg strains share more than 4,900 highly conserved genes, indicating very low genetic diversity between Psg genomes. Though conserved, genome rearrangements and recombination events occur commonly within the two Psg genomes. When compared to each other, 437 and 163 specific genes were identified in B076 and race 4, respectively. Most specific genes are plasmid-borne, indicating that acquisition and maintenance of plasmids may represent a major mechanism to change the genetic composition of the genome and even acquire new virulence factors. Type three secretion gene clusters of Psg strains are near identical with that of P. savastanoi pv. phaseolicola (Pph) strain 1448A and they shared 20 common effector genes. Furthermore, the coronatine biosynthetic cluster is present on a large plasmid in strain B076, but not in race 4. In silico subtractive hybridization-based comparative genomic analyses with nine sequenced phytopathogenic pseudomonads identified dozens of specific islands (SIs), and revealed that the genomes of Psg strains are more similar to those belonging to the same genomospecies such as Pph 1448A than to other phytopathogenic pseudomonads. The number of highly conserved genes (core genome) among them decreased dramatically when more genomes were included in the subtraction, suggesting the diversification of pseudomonads, and further indicating the genome heterogeneity among pseudomonads. However, the number of specific genes did not change significantly, suggesting these genes are indeed specific in Psg genomes. These results reinforce the idea of a species complex of P. syringae and support the reclassification of P. syringae into different species.

Comparative Genomics of Erwinia amylovora and Related Erwinia Species-What do We Learn?

Erwinia amylovora, the causal agent of fire blight disease of apples and pears, is one of the most important plant bacterial pathogens with worldwide economic significance. Recent reports on the complete or draft genome sequences of four species in the genus Erwinia, including E. amylovora, E. pyrifoliae, E. tasmaniensis, and E. billingiae, have provided us near complete genetic information about this pathogen and its closely-related species. This review describes in silico subtractive hybridization-based comparative genomic analyses of eight genomes currently available, and highlights what we have learned from these comparative analyses, as well as genetic and functional genomic studies. Sequence analyses reinforce the assumption that E. amylovora is a relatively homogeneous species and support the current classification scheme of E. amylovora and its related species. The potential evolutionary origin of these Erwinia species is also proposed. The current understanding of the pathogen, its virulence mechanism and host specificity from genome sequencing data is summarized. Future research directions are also suggested.

The Erwinia amylovora PhoPQ system is involved in resistance to antimicrobial peptide and suppresses gene expression of two novel type III secretion systems.

The PhoPQ system is a pleiotropic two-component signal transduction system that controls many pathogenic properties in several mammalian and plant pathogens. Three different cues have been demonstrated to activate the PhoPQ system including a mild acidic pH, antimicrobial peptides, and low Mg(2+). In this study, our results showed that phoPQ mutants were more resistant to strong acidic conditions (pH 4.5 or 5) than that of the wild-type (WT) strain, suggesting that this system in Erwinia amylovora may negatively regulate acid resistance gene expression. Furthermore, the PhoPQ system negatively regulated gene expression of two novel type III secretion systems in E. amylovora. These results are in contrast to those reported for the PhoPQ system in Salmonella and Xanthomonas, where it positively regulates type III secretion system and acid resistance. In addition, survival of phoPQ mutants was about 10-fold lower than that of WT when treated with cecropin A at pH 5.5, suggesting that the PhoPQ system renders the pathogen more resistant to cecropin A.

Comparative genomic and phylogenetic analyses reveal the evolution of the core two-component signal transduction systems in enterobacteria.

The two-component signal transduction system (TCST) consists of a histidine kinase (HK) and a response regulator (RR). TCSTs play important roles in sensing and reacting to environmental changes, and in bacterial pathogenesis. Previously, we have identified and characterized TCSTs in Erwinia amylovora, a severe plant enterobacterial pathogen, at genome-wide level. Here we conducted a comparative genomic analysis of TCSTs in 53 genomes of 16 enterobacterial species. These species include important plant, animal, human, and insect pathogenic, saprophytic or symbiotic microorganisms. Comparative genomic analysis revealed that enterobacteria contain eight pairs of core TCSTs. Phylogenetic trees reconstructed from a concatenation of the core set of TCSTs from enterobacteria and for individual TCST proteins from species in Proteobacteria showed that most TCST protein trees in the Enterobacteriaceae or in species of the γ-Proteobacteria agreed well with that of the corresponding 16S rRNA gene. It also showed that co-evolutionary relationships existed between cognate partners of the HKs and RRs. Several core TCSTs were quite ancient and universal based on phylogenomic analysis of protein structures. These results indicate that the core TCSTs are relatively conserved, and suggest that these enterobacteria may have maintained their ancient core TCSTs and might acquire specific new TCSTs for their survival in different environments or hosts, or may have evolved new functionalities of the core TCSTs for adaptation to different ecological niches.