CasPEDIA Database: a functional classification system for class 2 CRISPR-Cas enzymes.

CRISPR-Cas enzymes enable RNA-guided bacterial immunity and are widely used for biotechnological applications including genome editing. In particular, the Class 2 CRISPR-associated enzymes (Cas9, Cas12 and Cas13 families), have been deployed for numerous research, clinical and agricultural applications. However, the immense genetic and biochemical diversity of these proteins in the public domain poses a barrier for researchers seeking to leverage their activities. We present CasPEDIA (http://caspedia.org), the Cas Protein Effector Database of Information and Assessment, a curated encyclopedia that integrates enzymatic classification for hundreds of different Cas enzymes across 27 phylogenetic groups spanning the Cas9, Cas12 and Cas13 families, as well as evolutionarily related IscB and TnpB proteins. All enzymes in CasPEDIA were annotated with a standard workflow based on their primary nuclease activity, target requirements and guide-RNA design constraints. Our functional classification scheme, CasID, is described alongside current phylogenetic classification, allowing users to search related orthologs by enzymatic function and sequence similarity. CasPEDIA is a comprehensive data portal that summarizes and contextualizes enzymatic properties of widely used Cas enzymes, equipping users with valuable resources to foster biotechnological development. CasPEDIA complements phylogenetic Cas nomenclature and enables researchers to leverage the multi-faceted nucleic-acid targeting rules of diverse Class 2 Cas enzymes.

Transcriptome and Comparative Genomics Analyses Reveal New Functional Insights on Key Determinants of Pathogenesis and Interbacterial Competition in Pectobacterium and Dickeya spp.

Soft-rot Enterobacteriaceae (SRE), typified by Pectobacterium and Dickeya genera, are phytopathogenic bacteria inflicting soft-rot disease in crops worldwide. By combining genomic information from 100 SRE with whole-transcriptome data sets, we identified novel genomic and transcriptional associations among key pathogenicity themes in this group. Comparative genomics revealed solid linkage between the type I secretion system (T1SS) and the carotovoricin bacteriophage (Ctv) conserved in 96.7% of Pectobacterium genomes. Moreover, their coactivation during infection indicates a novel functional association involving T1SS and Ctv. Another bacteriophage-borne genomic region, mostly confined to less than 10% of Pectobacterium strains, was found, presumably comprising a novel lineage-specific prophage in the genus. We also detected the transcriptional coregulation of a previously predicted toxin/immunity pair (WHH and SMI1_KNR4 families), along with the type VI secretion system (T6SS), which includes hcp and/or vgrG genes, suggesting a role in disease development as T6SS-dependent effectors. Further, we showed that another predicted T6SS-dependent endonuclease (AHH family) exhibited toxicity in ectopic expression assays, indicating antibacterial activity. Additionally, we report the striking conservation of the group 4 capsule (GFC) cluster in 100 SRE strains which consistently features adjacently conserved serotype-specific gene arrays comprising a previously unknown organization in GFC clusters. Also, extensive sequence variations found in gfcA orthologs suggest a serotype-specific role in the GfcABCD machinery.IMPORTANCE Despite the considerable loss inflicted on important crops yearly by Pectobacterium and Dickeya diseases, investigations on key virulence and interbacterial competition assets relying on extensive comparative genomics are still surprisingly lacking for these genera. Such approaches become more powerful over time, underpinned by the growing amount of genomic information in public databases. In particular, our findings point to new functional associations among well-known genomic themes enabling alternative means of neutralizing SRE diseases through disruption of pivotal virulence programs. By elucidating novel transcriptional and genomic associations, this study adds valuable information on virulence candidates that could be decisive in molecular applications in the near future. The utilization of 100 genomes of Pectobacterium and Dickeya strains in this study is unprecedented for comparative analyses in these taxa, and it provides novel insights on the biology of economically important plant pathogens.

Expansion and diversification of the gibberellin receptor GIBBERELLIN INSENSITIVE DWARF1 (GID1) family in land plants.

KEY MESSAGE: Here we uncover the major evolutionary events shaping the evolution of the GID1 family of gibberellin receptors in land plants at the sequence, structure and gene expression levels. Gibberellic acid (gibberellin, GA) controls key developmental processes in the life cycle of land plants. By interacting with the GIBBERELLIN INSENSITIVE DWARF1 (GID1) receptor, GA regulates the expression of a wide range of genes through different pathways. Here we report the systematic identification and classification of GID1s in 54 plants genomes, encompassing from bryophytes and lycophytes, to several monocots and eudicots. We investigated the evolutionary relationship of GID1s using a comparative genomics framework and found strong support for a previously proposed phylogenetic classification of this family in land plants. We identified lineage-specific expansions of particular subfamilies (i.e. GID1ac and GID1b) in different eudicot lineages (e.g. GID1b in legumes). Further, we found both, shared and divergent structural features between GID1ac and GID1b subgroups in eudicots that provide mechanistic insights on their functions. Gene expression data from several species show that at least one GID1 gene is expressed in every sampled tissue, with a strong bias of GID1b expression towards underground tissues and dry legume seeds (which typically have low GA levels). Taken together, our results indicate that GID1ac retained canonical GA signaling roles, whereas GID1b specialized in conditions of low GA concentrations. We propose that this functional specialization occurred initially at the gene expression level and was later fine-tuned by mutations that conferred greater GA affinity to GID1b, including a Phe residue in the GA-binding pocket. Finally, we discuss the importance of our findings to understand the diversification of GA perception mechanisms in land plants.

A map of the rubisco biochemical landscape.

Rubisco is the primary CO2 fixing enzyme of the biosphere yet has slow kinetics. The roles of evolution and chemical mechanism in constraining the sequence landscape of rubisco remain debated. In order to map sequence to function, we developed a massively parallel assay for rubisco using an engineered E. coli where enzyme function is coupled to growth. By assaying >99% of single amino acid mutants across CO2 concentrations, we inferred enzyme velocity and CO2 affinity for thousands of substitutions. We identified many highly conserved positions that tolerate mutation and rare mutations that improve CO2 affinity. These data suggest that non-trivial kinetic improvements are readily accessible and provide a comprehensive sequence-to-function mapping for enzyme engineering efforts.

Novel Two-Component System-Like Elements Reveal Functional Domains Associated with Restriction-Modification Systems and paraMORC ATPases in Bacteria.

Two-component systems (TCS) are important types of machinery allowing for efficient signal recognition and transmission in bacterial cells. The majority of TCSs utilized by bacteria is composed of a sensor histidine kinase (HK) and a cognate response regulator (RR). In the present study, we report two newly predicted protein domains-both to be included in the next release of the Pfam database: Response_reg_2 (PF19192) and HEF_HK (PF19191)-in bacteria which exhibit high structural similarity, respectively, with typical domains of RRs and HKs. Additionally, the genes encoding for the novel predicted domains exhibit a 91.6% linkage observed across 644 genomic regions recovered from 628 different bacterial strains. The remarkable adjacent colocalization between genes carrying Response_reg_2 and HEF_HK in addition to their conserved structural features, which are highly similar to those from well-known HKs and RRs, raises the possibility of Response_reg_2 and HEF_HK constituting a new TCS in bacteria. The genomic regions in which these predicted two-component systems-like are located additionally exhibit an overrepresented presence of restriction-modification (R-M) systems especially the type II R-M. Among these, there is a conspicuous presence of C-5 cytosine-specific DNA methylases which may indicate a functional association with the newly discovered domains. The solid presence of R-M systems and the presence of the GHKL family domain HATPase_c_3 across most of the HEF_HK-containing genes are also indicative that these genes are evolutionarily related to the paraMORC family of ATPases.

Pectobacterium brasiliense 1692 Chemotactic Responses and the Role of Methyl-Accepting Chemotactic Proteins in Ecological Fitness.

To adapt to changing environmental niches, bacteria require taxis, a movement toward or away from a stimulus (ligand). Chemotaxis has been studied in some members of the Soft Rot Pectobacteriaceae (SRP), particularly members of the genus Dickeya. On the contrary, there are fewer studies on this topic for the other genus in the SRP group, namely Pectobacterium. This study evaluated chemotactic responses in Pectobacterium brasiliense (Pb 1692) to various ligands. A total of 34 methyl-accepting chemotactic proteins (MCPs) were identified in the Pb 1692 genome and the domain architectures of these MCPs were determined. Four Pb 1692 MCPs previously shown to be differentially expressed during potato tuber infection were selected for further functional characterization. Toward this end, Pb 1692 mutant strains each lacking either AED-0001492, AED-0003671, AED-0000304, or AED-0000744 were generated. Two of these mutants (AED-0001492 and AED-0003671), were attenuated in their ability to grow and respond to citrate and are thus referred to as MCP cit2 and MCP cit1 , respectively, while the other two, AED-0000304 (MCP xyl ) and AED-0000744 (MCP asp ), were affected in their ability to respond to xylose and aspartate, respectively. Trans-complementation of the mutant strains restored swimming motility in the presence of respective ligands. The four MCP mutants were not affected in virulence but were significantly attenuated in their ability to attach to potato leaves suggesting that ecological fitness is an important contribution of these MCPs toward Pb 1692 biology.

Transcriptome Profiling of Potato (Solanum tuberosum L.) Responses to Root-Knot Nematode (Meloidogyne javanica) Infestation during A Compatible Interaction.

Root-knot nematode (RKN) Meloidogyne javanica presents a great challenge to Solanaceae crops, including potato. In this study, we investigated transcriptional responses of potato roots during a compatible interaction with M. javanica. In this respect, differential gene expression of Solanum tuberosum cultivar (cv.) Mondial challenged with M. javanica at 0, 3 and 7 days post-inoculation (dpi) was profiled. In total, 4948 and 4484 genes were detected, respectively, as differentially expressed genes (DEGs) at 3 and 7 dpi. Functional annotation revealed that genes associated with metabolic processes were enriched, suggesting they might have an important role in M. javanica disease development. MapMan analysis revealed down-regulation of genes associated with pathogen perception and signaling suggesting interference with plant immunity system. Notably, delayed activation of pathogenesis-related genes, down-regulation of disease resistance genes, and activation of host antioxidant system contributed to a susceptible response. Nematode infestation suppressed ethylene (ET) and jasmonic acid (JA) signaling pathway hindering JA/ET responsive genes associated with defense. Genes related to cell wall modification were differentially regulated while transport-related genes were up-regulated, facilitating the formation of nematode feeding sites (NFSs). Several families of transcription factors (TFs) were differentially regulated by M. javanica infestation. Suggesting that TFs play an indispensable role in physiological adaptation for successful M. javanica disease development. This genome-wide analysis reveals the molecular regulatory networks in potato roots which are potentially manipulated by M. javanica. Being the first study analyzing transcriptome profiling of M. javanica-diseased potato, it provides unparalleled insight into the mechanism underlying disease development.

Organize, Don't Agonize: Strategic Success of Phytophthora Species.

Plants are constantly challenged by various environmental stressors ranging from abiotic-sunlight, elevated temperatures, drought, and nutrient deficits, to biotic factors-microbial pathogens and insect pests. These not only affect the quality of harvest but also the yield, leading to substantial annual crop losses, worldwide. Although plants have a multi-layered immune system, phytopathogens such as species of the oomycete genus Phytophthora, can employ elaborate mechanisms to breach this defense. For the last two decades, researchers have focused on the co-evolution between Phytophthora and interacting hosts to decouple the mechanisms governing their molecular associations. This has provided a comprehensive understanding of the pathobiology of plants affected by oomycetes. Ultimately, this is important for the development of strategies to sustainably improve agricultural production. Therefore, this paper discusses the present-day state of knowledge of the strategic mode of operation employed by species of Phytophthora for successful infection. Specifically, we consider motility, attachment, and host cell wall degradation used by these pathogenic species to obtain nutrients from their host. Also discussed is an array of effector types from apoplastic (hydrolytic proteins, protease inhibitors, elicitins) to cytoplastic (RxLRs, named after Arginine-any amino acid-Leucine-Arginine consensus sequence and CRNs, for CRinkling and Necrosis), which upon liberation can subvert the immune response and promote diseases in plants.

Horizontally Acquired Quorum-Sensing Regulators Recruited by the PhoP Regulatory Network Expand the Host Adaptation Repertoire in the Phytopathogen Pectobacterium brasiliense.

In this study, we examine the impact of transcriptional network rearrangements driven by horizontal gene acquisition in PhoP and SlyA regulons using as a case study a phytopathosystem comprised of potato tubers and the soft-rot pathogen Pectobacterium brasiliense 1692 (Pb1692). Genome simulations and statistical analyses uncovered the tendency of PhoP and SlyA networks to mobilize lineage-specific traits predicted as horizontal gene transfer at late infection, highlighting the prominence of regulatory network rearrangements in this stage of infection. The evidence further supports the circumscription of two horizontally acquired quorum-sensing regulators (carR and expR1) by the PhoP network. By recruiting carR and expR1, the PhoP network also impacts certain host adaptation- and bacterial competition-related systems, seemingly in a quorum sensing-dependent manner, such as the type VI secretion system, carbapenem biosynthesis, and plant cell wall-degrading enzymes (PCWDE) like cellulases and pectate lyases. Conversely, polygalacturonases and the type III secretion system (T3SS) exhibit a transcriptional pattern that suggests quorum-sensing-independent regulation by the PhoP network. This includes an uncharacterized novel phage-related gene family within the T3SS gene cluster that has been recently acquired by two Pectobacterium species. The evidence further suggests a PhoP-dependent regulation of carbapenem- and PCWDE-encoding genes based on the synthesized products' optimum pH. The PhoP network also controls slyA expression in planta, which seems to impact carbohydrate metabolism regulation, especially at early infection, when 76.2% of the SlyA-regulated genes from that category also require PhoP to achieve normal expression levels.IMPORTANCE Exchanging genetic material through horizontal transfer is a critical mechanism that drives bacteria to efficiently adapt to host defenses. In this report, we demonstrate that a specific plant-pathogenic species (from the Pectobacterium genus) successfully integrated a population density-based behavior system (quorum sensing) acquired through horizontal transfer into a resident stress-response gene regulatory network controlled by the PhoP protein. Evidence found here underscores that subsets of bacterial weaponry critical for colonization, typically known to respond to quorum sensing, are also controlled by PhoP. Some of these traits include different types of enzymes that can efficiently break down plant cell walls depending on the environmental acidity level. Thus, we hypothesize that PhoP's ability to elicit regulatory responses based on acidity and nutrient availability fluctuations has strongly impacted the fixation of its regulatory connection with quorum sensing. In addition, another global gene regulator, known as SlyA, was found under the PhoP regulatory network. The SlyA regulator controls a series of carbohydrate metabolism-related traits, which also seem to be regulated by PhoP. By centralizing quorum sensing and slyA under PhoP scrutiny, Pectobacterium cells added an advantageous layer of control over those two networks that potentially enhances colonization efficiency.

The Impact of Type VI Secretion System, Bacteriocins and Antibiotics on Bacterial Competition of Pectobacterium carotovorum subsp. brasiliense and the Regulation of Carbapenem Biosynthesis by Iron and the Ferric-Uptake Regulator.

The complexity of plant microbial communities provides a rich model for investigating biochemical and regulatory strategies involved in interbacterial competition. Within these niches, the soft rot Enterobacteriaceae (SRE) represents an emerging group of plant-pathogens causing soft rot/blackleg diseases resulting in economic losses worldwide in a variety of crops. A preliminary screening using next-generation sequencing of 16S rRNA comparatively analyzing healthy and diseased potato tubers, identified several taxa from Proteobacteria to Firmicutes as potential potato endophytes/plant pathogens. Subsequent to this, a range of molecular and computational techniques were used to determine the contribution of antimicrobial factors such as bacteriocins, carbapenem and type VI secretion system (T6SS), found in an aggressive SRE (Pectobacterium carotovorum subsp. brasiliense strain PBR1692 - Pcb1692) against these endophytes/plant pathogens. The results showed growth inhibition of several Proteobacteria by Pcb1692 depends either on carbapenem or pyocin production. Whereas for targeted Firmicutes, only the Pcb1692 pyocin seems to play a role in growth inhibition. Furthermore, production of carbapenem by Pcb1692 was observably dependent on the presence of environmental iron and oxygen. Additionally, upon deletion of fur, slyA and expI regulators, carbapenem production ceased, implying a complex regulatory mechanism involving these three genes. Finally, the results demonstrated that although T6SS confers no relevant advantage during in vitro competition, a significant attenuation in competition by the mutant strain lacking a functional T6SS was observed in planta. IMPORTANCE: Soft rot Enterobacteriaceae (SRE) represents important phytopathogens causing soft rot/blackleg diseases in a variety of crops leading to huge economic losses worldwide. These pathogens have been isolated alongside other bacteria from different environments such as potato tubers, stems, roots and from the soil. In these environments, SREs coexist with other bacteria where they have to compete for scarce nutrients and other resources. In this report, we show that Pectobacterium carotovorum subsp. brasiliense strain PBR1692 - Pcb1692, which represents one of the SREs, inhibits growth of several different bacteria by producing different antimicrobial compounds. These antimicrobial compounds can be secreted inside or outside the plant host, allowing Pcb1692 to effectively colonize different types of ecological niches. By analyzing the genome sequences of several SREs, we show that other SREs likely deploy similar antimicrobials to target other bacteria.

De novo transcriptome sequencing and comparative analysis of midgut tissues of four non-model insects pertaining to Hemiptera, Coleoptera, Diptera and Lepidoptera.

Despite the great morphological diversity of insects, there is a regularity in their digestive functions, which is apparently related to their physiology. In the present work we report the de novo midgut transcriptomes of four non-model insects from four distinct orders: Spodoptera frugiperda (Lepidoptera), Musca domestica (Diptera), Tenebrio molitor (Coleoptera) and Dysdercus peruvianus (Hemiptera). We employed a computational strategy to merge assemblies obtained with two different algorithms, which substantially increased the quality of the final transcriptomes. Unigenes were annotated and analyzed using the eggNOG database, which allowed us to assign some level of functional and evolutionary information to 79.7% to 93.1% of the transcriptomes. We found interesting transcriptional patterns, such as: i) the intense use of lysozymes in digestive functions of M. domestica larvae, which are streamlined and adapted to feed on bacteria; ii) the up-regulation of orthologous UDP-glycosyl transferase and cytochrome P450 genes in the whole midguts different species, supporting the existence of an ancient defense frontline to counter xenobiotics; iii) evidence supporting roles for juvenile hormone binding proteins in the midgut physiology, probably as a way to activate genes that help fight anti-nutritional substances (e.g. protease inhibitors). The results presented here shed light on the digestive and structural properties of the digestive systems of these distantly related species. Furthermore, the produced datasets will also be useful for scientists studying these insects.

Transcriptome analysis uncovers key regulatory and metabolic aspects of soybean embryonic axes during germination.

Soybean (Glycine max) is a major legume crop worldwide, providing a critical source of protein and oil. The release of the soybean genome fuelled several transcriptome projects comprising multiple developmental stages and environmental conditions. Nevertheless, the global transcriptional patterns of embryonic axes during germination remain unknown. Here we report the analysis of ~1.58 billion RNA-Seq reads from soybean embryonic axes at five germination stages. Our results support the early activation of processes that are critical for germination, such as glycolysis, Krebs cycle and cell wall remodelling. Strikingly, only 3 hours after imbibition there is a preferential up-regulation of protein kinases and transcription factors, particularly from the LOB domain family, implying that transcriptional and post-transcriptional regulation play major roles early after imbibition. Lipid mobilization and glyoxylate pathways are also transcriptionally active in the embryonic axes, indicating that the local catabolism of oil reserves in the embryonic axes contributes to energy production during germination. We also present evidence supporting abscisic acid inactivation and the up-regulation of gibberellin, ethylene and brassinosteroid pathways. Further, there is a remarkable differential activation of paralogous genes in these hormone signalling pathways. Taken together, our results provide insights on the regulation and biochemistry of soybean germination.

Evolutionary analysis of multidrug resistance genes in fungi - impact of gene duplication and family conservation.

Although the emergence of bacterial drug resistance is of great concern to the scientific community, few studies have evaluated this phenomenon systematically in fungi by using genome-wide datasets. In the present study, we assembled a large compendium of Saccharomyces cerevisiae chemical genetic data to study the evolution of multidrug resistance genes (MDRs) in the fungal lineage. We found that MDRs typically emerge in widely conserved families, most of which containing homologs from pathogenic fungi, such as Candida albicans and Coccidioides immitis, which could favor the evolution of drug resistance in those species. By integrating data from chemical genetics with protein family conservation, genetic and protein interactions, we found that gene families rarely have more than one MDR, indicating that paralogs evolve asymmetrically with regard to multidrug resistance roles. Furthermore, MDRs have more genetic and protein interaction partners than non-MDRs, supporting their participation in complex biochemical systems underlying the tolerance to multiple bioactive molecules. MDRs share more chemical genetic interactions with other MDRs than with non-MDRs, regardless of their evolutionary affinity. These results suggest the existence of an intricate system involved in the global drug tolerance phenotypes. Finally, MDRs are more likely to be hit repeatedly by mutations in laboratory evolution experiments, indicating that they have great adaptive potential. The results presented here not only reveal the main genomic features underlying the evolution of MDRs, but also shed light on the gene families from which drug resistance is more likely to emerge in fungi.

Impact of whole-genome and tandem duplications in the expansion and functional diversification of the F-box family in legumes (Fabaceae).

F-box proteins constitute a large gene family that regulates processes from hormone signaling to stress response. F-box proteins are the substrate recognition modules of SCF E3 ubiquitin ligases. Here we report very distinct trends in family size, duplication, synteny and transcription of F-box genes in two nitrogen-fixing legumes, Glycine max (soybean) and Medicago truncatula (alfafa). While the soybean FBX genes emerged mainly through segmental duplications (including whole-genome duplications), M. truncatula genome is dominated by locally-duplicated (tandem) F-box genes. Many of these young FBX genes evolved complex transcriptional patterns, including preferential transcription in different tissues, suggesting that they have probably been recruited to important biochemical pathways (e.g. nodulation and seed development).

Evolutionary and Biochemical Aspects of Chemical Stress Resistance in Saccharomyces cerevisiae.

Large-scale chemical genetics screens (chemogenomics) in yeast have been widely used to find drug targets, understand the mechanism-of-action of compounds, and unravel the biochemistry of drug resistance. Chemogenomics is based on the comparison of growth of gene deletants in the presence and absence of a chemical substance. Such studies showed that more than 90% of the yeast genes are required for growth in the presence of at least one chemical. Analysis of these data, using computational approaches, has revealed non-trivial features of the natural chemical tolerance systems. As a result two non-overlapping sets of genes are seen to respectively impart robustness and evolvability in the context of natural chemical resistance. The former is composed of multidrug-resistance genes, whereas the latter comprises genes sharing chemical genetic profiles with many others. Recent publications showing the potential applications chemogenomics in studying the pharmacological basis of various drugs are discussed, as well as the expansion of chemogenomics to other organisms. Finally, integration of chemogenomics with sensitive sequence analysis and ubiquitination/phosphorylation data led to the discovery of a new conserved domain and important post-translational modification pathways involved in stress resistance.