Bacterial expansins contribute to the virulence of diverse plant pathogens, yet their biological activity and molecular roles during host interactions remain to be elucidated. Expansin-like protein, Exl1, from phytopathogens from the genus Pectobacterium, activates defense responses in Arabidopsis thaliana involving production of reactive oxygen species (ROS), and the jasmonic acid (JA), ethylene (ET), and salicylic acid (SA) pathways, ultimately reducing disease symptoms upon subsequent infections with P. brasiliense and the fungus Botrytis cinerea. However, the target of Exl1 within the plant cell wall and the downstream pathways associated with these responses are yet to be identified. Here, we analyzed the transcriptomic profile of A. thaliana leaves treated with Exl1 protein to provide a genome-wide transcriptional framework associated with Exl1-triggered immunity. Comparative transcriptomic analysis of Arabidopsis thaliana Col-0 and JA-impaired mutant jar1-1 revealed distinct transcriptional patterns associated with JA-dependent responses at early time points. Abscisic acid (ABA)-related signaling was associated with later stages of the response, supported by the increased expression of the ABA-responsive gene CRK45 six hours after Exl1 treatment, and increased susceptibility of an ABA-impaired mutant. These findings suggest the involvement of multiple phytohormone-related pathways following Exl1 treatment. In parallel, extensive transcriptional reprogramming was observed, across diverse functional categories, reflecting a broad host response associated to the presence of Exl1. Together, these findings suggest that Exl1 induces an integrated signaling network that coordinates immune responses to strengthen plant defense, providing new insight for future functional studies on how bacterial expansins modulate plant-microbe interactions.

Parasites can counteract host immunity by suppressing nucleotide binding and leucine-rich repeat (NLR) proteins that function as immune receptors. We previously showed that a cyst nematode virulence effector SPRYSEC15 (SS15) binds and inhibits oligomerisation of helper NLR proteins in the expanded NRC1/2/3 clade by preventing intramolecular rearrangements required for NRC oligomerisation into an activated resistosome. Here we examined the degree to which NRC proteins from multiple Solanaceae species are sensitive to suppression by SS15 and tested hypotheses about adaptive evolution of the binding interface between the SS15 inhibitor and NRC proteins. Whereas all tested orthologs of NRC2 were inhibited by SS15, some natural variants of NRC1 and NRC3 are insensitive to SS15 suppression. Ancestral sequence reconstruction combined with functional assays revealed that NRC3 transitioned from an ancestral suppressed form to an insensitive one over 19 million years ago. Our analyses revealed the evolutionary trajectory of an NLR immune receptor against a parasite inhibitor, identifying key evolutionary transitions in helper NLRs that counteract this inhibition. This work reveals a distinct type of gene-for-gene interaction between parasite or pathogen immunosuppressors and host immune receptors that contrasts with the coevolution between AVR effectors and immune receptors.

Abstract M. enterolobii is an emergent phytopathogenic nematode that infects and reduces the yields of commercially important vegetables, such as tomatoes (Solanum lycopersicum). In Mexico, M. enterolobii has gained access to states previously unknown to be affected by it, especially northern states such as Sinaloa, which is the main producer of tomato in the country. Formulations based on antagonistic microorganisms exist in the Mexican market and are used in combination with chemically synthesized pesticides, which although effective are also unspecific and even toxic to other animals and humans. The ascomycetes Purpureocillum lilacinum (previously, Paecilomyces lilacinus) is an effective nematicide already used in different commercial formulations. Here, we aimed to determine the combined effect of an environmental strain of P. lilacinum MTL01 that we isolated, with spores of B. velezensis 83 that is also a growth promoter of tomato. Our findings of two assays in greenhouse conditions of tomato development treated with MTL01 and B. velezensis 83, either individually or in combination, and then challenged with stage-2 juveniles (J2) of M. enterolobii, indicate that the combined treatment is useful for reducing galling and stimulating flowering, with the potential of better yields to produce greenhouse-grown tomato.

ABSTRACT NLR (nucleotide-binding domain and leucine-rich repeat-containing) proteins can form complex receptor networks to confer innate immunity. NRCs are phylogenetically related nodes that function downstream of a massively expanded network of disease resistance proteins that protect against multiple plant pathogens. Here, we used phylogenomic methods to reconstruct the macroevolution of the NRC family. One of the NRCs, we termed NRC0 , is the only family member shared across asterid plants, leading us to investigate its evolutionary history and genetic organization. In several asterid species, NRC0 is genetically clustered to other NLRs that are phylogenetically related to NRC-dependent disease resistance genes. This prompted us to hypothesize that the ancestral state of the NRC network is an NLR helper-sensor gene cluster that was present early during asterid evolution. We validated this hypothesis by demonstrating that NRC0 is essential for the hypersensitive cell death induced by its genetically linked sensor NLR partners in four divergent asterid species: tomato, wild sweet potato, coffee and carrot. In addition, activation of a sensor NLR leads to high-order complex formation of its genetically linked NRC0 similar to other NRCs. Our findings map out contrasting evolutionary dynamics in the macroevolution of the NRC network over the last 125 million years from a functionally conserved NLR gene cluster to a massive genetically dispersed network. One-sentence summary NRC0 gene cluster is functionally conserved across divergent asterid species and predates the massively expanded NRC network.

The bacterial cell envelope is involved in all stages of infection and the study of its components and structures is important to understand how bacteria interact with the extracellular milieu. Thanks to new techniques that focus on identifying bacterial surface proteins, we now better understand the specific components involved in host–pathogen interactions. In the fight against the deleterious effects of pathogenic bacteria, bacterial surface proteins (at the cell envelope) are important targets as they play crucial roles in the colonization and infection of host tissues. These surface proteins serve functions such as protection, secretion, biofilm formation, nutrient intake, metabolism, and virulence. Bacteria use different mechanisms to associate proteins to the cell surface via posttranslational modification, such as the addition of a lipid moiety to create lipoproteins and attachment to the peptidoglycan layer by sortases. In this review, we focus on these types of proteins (and provide examples of others) that are associated with the bacterial cell envelope by posttranslational modifications and their roles in plant infection.

Expansins are a group of proteins from diverse organisms from bacteria to plants. Although expansins show structural conservation, their biological roles seem to differ among kingdoms. In plants, these proteins remodel the cell wall during plant growth and other processes. Contrarily, determination of bacterial expansin activity has proven difficult, although genetic evidence of bacterial mutants indicates that expansins participate in bacteria-plant interactions. Nevertheless, a large proportion of expansin genes are found in the genomes of free-living bacteria, suggesting roles that are independent of the interaction with living plants. Here, we analyzed all available sequences of prokaryotic expansins for correlations between surface electric charge, extra protein modules, and sequence motifs for association with the bacteria exterior after export. Additionally, information on the fate of protein after translocation across the membrane also points to bacterial cell association of expansins through six different mechanisms, such as attachment of a lipid molecule for membrane anchoring in diderm species or covalent linking to the peptidoglycan layer in monoderms such as the Bacilliales. Our results have implications for expansin function in the context of bacteria-plant interactions and also for free-living species in which expansins might affect cell-cell or cell-substrate interaction properties and indicate the need to re-examine the roles currently considered for these proteins.

Cell wall integrity is tightly regulated and maintained given that non-physiological modification of cell walls could render plants vulnerable to biotic and/or abiotic stresses. Expansins are plant cell wall-modifying proteins active during many developmental and physiological processes, but they can also be produced by bacteria and fungi during interaction with plant hosts. Cell wall alteration brought about by ectopic expression, overexpression, or exogenous addition of expansins from either eukaryote or prokaryote origin can in some instances provide resistance to pathogens, while in other cases plants become more susceptible to infection. In these circumstances altered cell wall mechanical properties might be directly responsible for pathogen resistance or susceptibility outcomes. Simultaneously, through membrane receptors for enzymatically released cell wall fragments or by sensing modified cell wall barrier properties, plants trigger intracellular signaling cascades inducing defense responses and reinforcement of the cell wall, contributing to various infection phenotypes, in which expansins might also be involved. Here, we review the plant immune response activated by cell wall surveillance mechanisms, cell wall fragments identified as responsible for immune responses, and expansin's roles in resistance and susceptibility of plants to pathogen attack.

Root-knot nematodes (RKN) are sedentary parasites of the roots of plants and are considered some of the most damaging pests in agriculture. Since RKN target the root vascular system, they provoke host nutrient deprivation and defective water transport, causing above-ground symptoms of growth stunting, wilting, chlorosis, and reduced crop yields. In Mexico RKN infestations are primarily dealt with by treating with synthetic chemically based nematicides that are preferred by farmers over available bioproducts. However, due to environmental and human health concerns chemical control is increasingly restricted. Biological control of RKNs can help reduce the use of chemical nematicides as it is achieved with antagonistic organisms, mainly bacteria, fungi, other nematodes, or consortia of diverse microorganisms, which control nematodes directly by predation and parasitism at different stages: eggs, juveniles, or adults; or indirectly by the action of toxic diffusible inhibitory metabolites. The need to increase agricultural production and reduce negative environmental impact creates an opportunity for optimizing biological control agents to suppress nematode populations, but this endeavour remains challenging as researchers around the world try to understand diverse control mechanisms, nematode and microbe life cycles, ecology, metabolite production, predatory behaviours, molecular and biochemical interactions, in order to generate attractive products with the approval of local regulatory bodies. Here, we provide a brief review of the biology of the genus Meloidogyne, biological control strategies, and a comparison between chemical and bioproducts in the Mexican market, and guidelines emitted by national agencies to ensure safety and effectiveness of new developments.

Neobuxbaumia tetetzo , commonly known as tetecho, is a columnar cactus endemic to Mexico. In the last 15 years, damage has been observed in young and adult plants of N. tetetzo , ranging from chlorotic spots with a wet appearance in early stages to tissue necrosis in advanced stages and finally the death of the plant; Pectobacterium brasiliense is the causal agent of the damages. Disease progression may be delayed or accelerated by the involvement of other bacteria, either pathogenic or endophytic, at the site of infection. Our goal was to confirm the presence of Pectobacterium brasiliense , in the soft rot of N. tetetzo and to determine the presence of other bacteria associated with the rot. We isolated three bacterial strains (A1, A3 and A8) from diseased tissue from three separate N. tetetzo plants, and compare them using biochemical and molecular techniques, such as whole-genome sequencing of strains A1 and A3. Phylogenetic analyzes confirmed that A1 corresponded to P. brasiliense , whereas A3 was more misimlar to P. polaris . Additionally, sequencing of 16S rRNA gene from metagenomic DNA isolated from healthy and diseased tissue of N. tetetzo indicated the presence of four operational taxonomic units (OTUs) at the order level, unique to the diseased tissue: Actinomycetales, Burkholderiales, Caulobacterales, and Sphingomonadales, with probable participation in the soft rot process.

Expansins, cerato-platanins and swollenins (which we will henceforth refer to as expansin-related proteins) are a group of microbial proteins involved in microbe- plant interactions. Although they share very low sequence similarity, some of their composing domains are near-identical at the structural level. Expansin-related proteins have their target in the plant cell wall, in which they act through a non- enzymatic, but still uncharacterized, mechanism. In most cases, mutagenesis of expansin-related genes affects plant colonization or plant pathogenesis of different bacterial and fungal species, and thus, in many cases they are considered virulence factors. Additionally, plant treatment with expansin-related proteins activate several plant defenses resulting in the priming and protection towards subsequent pathogen encounters. Plant-defence responses induced by these proteins are reminiscent of pattern-triggered immunity or hypersensitive response in some cases. Plant immunity to expansin-related proteins could be caused by the following: (i) protein detection by specific host-cell receptors, (ii) alterations to the cell-wall-barrier properties sensed by the host, (iii) displacement of cell-wall polysaccharides detected by the host. Expansin-related proteins may also target polysaccharides on the wall of the microbes that produced them under certain physiological instances. Here, we review biochemical, evolutionary and biological aspects of these relatively understudied proteins and different immune responses they induce in plant hosts.

Expansins are encoded by some phytopathogenic bacteria and evidence indicates that they act as virulence factors for host infection. Here we analysed the expression of exl1 by Pectobacterium brasiliense and Pectobacterium atrosepticum . In both, exl1 gene appears to be under quorum sensing control, and protein Exl1 can be observed in culture medium and during plant infection. Expression of exl1 correlates with pathogen virulence, where symptoms are reduced in a Δexl1 mutant strain of P. atrosepticum . As well as Δexl1 exhibiting less maceration of potato plants, fewer bacteria are observed at distance from the inoculation site. However, bacteria infiltrated into the plant tissue are as virulent as the wild type, suggesting that this is due to alterations in the initial invasion of the tissue. Additionally, swarming from colonies grown on MacConkey soft agar was delayed in the mutant in comparison to the wild type. We found that Exl1 acts on the plant tissue, probably by remodelling of a cell wall component or altering the barrier properties of the cell wall inducing a plant defence response, which results in the production of ROS and the induction of marker genes of the JA, ET and SA signalling pathways in Arabidopsis thaliana . Exl1 inactive mutants fail to trigger such responses. This defence response is protective against Pectobacterium brasiliense and Botrytis cinerea in more than one plant species.

El inicio de la década de los 20 no fue normal. Nos sorprendió con una noticia grande, de interés para todos, de fuera de México y posteriormente también aquí. El año 2020 inició con la noticia de que en la ciudad de Wuhan en China, había un brote de neumonías virales que provocaba que la gente buscara atención médica y cuya evolución no era igual a las infecciones respiratorias agudas conocidas. Uno de los primeros médicos, el oftalmólogo Li Wenliang notó el alto número de pacientes con síntomas parecidos y en diciembre del 2019 comentó que la enfermedad podría ser causada por un virus nuevo. El 7 de febrero del 2020 el Dr. Wenliang había muerto por la constante exposición a tal virus. Diez días después siguieron las muertes de otros médicos, el Dr. Liu Zhiming y el Dr. Xu Depu quien fuera director del hospital médico de la ciudad Ezhou en Hubei, además de muchos pacientes. Desde entonces, y hasta el 24 de marzo hay casi 400,000 infecciones y cerca de 20,000 muertos en todo el mundo, sin que parezca que los contagios comiencen a disminuir. Pero ¿qué es lo que ocurre cuando el virus infecta? ¿Por qué es importante saber si alguien está infectado? ¿En qué consiste la prueba clínica? ¿Qué tipo de vigilancia epidemiológica se lleva a cabo en México? En este artículo se contestamos estas preguntas.

Pectobacterium is a diverse genus of phytopathogenic species from soil and water that cause infection either to restricted or multiple plant hosts. Phylogenetic analysis and metabolic fingerprinting of large numbers of genomes have expanded classification of Pectobacterium members. Pectobacterium brasiliense sp. nov has been elevated to the species level having detached from P. carotovorum. Here we present two P. brasiliense strains BF20 and BF45 isolated in Mexico from Opuntia and tobacco, respectively, which cluster into two different groups in whole genome comparisons with other Pectobacterium. We found that BF20 and BF45 strains are phenotypically different as BF45 showed more severe and rapid symptoms in comparison to BF20 in the host models celery and broccoli. Both strains produced similar levels of the main autoinducers, but BF45 shows an additional low abundant autoinducer compared to strain BF20. The two strains had different levels of c-di-GMP, which regulates the transition from motile to sessile lifestyle. In contrast to BF45, BF20 had the highest levels of c-di-GMP, was more motile (swarming), non-flocculant and less proficient in biofilm formation and exopolysaccharide production. Genomic comparisons revealed that differences in c-di-GMP accumulation and perhaps the associated phenotypes might be due to unique c-di-GMP metabolic genes in these two strains. Our results improve our understanding of the associations between phenotype and genotype and how this has shaped the physiology of Pectobacterium strains.

The plant xylem is a preferred niche for some important bacterial phytopathogens, some of them encoding expansin proteins, which bind plant cell walls. Yet, the identity of the substrate for bacterial expansins within the plant cell wall and the nature of its interaction with it are poorly known. Here, we determined the localization of two bacterial expansins with differing isoelectric points (and with differing binding patterns to cell wall extracts) on plant tissue through in vitro fluorophore labeling and confocal imaging. Differential localization was observed, in which Exl1 from Pectobacterium carotovorum located into the intercellular spaces between xylem vessels and adjacent cells of the plant xylem, whereas EXLX1 from Bacillus subtilis bound cell walls of most cell types. In isolated vascular tissue, however, both PcExl1 and BsEXLX1 preferentially bound to tracheary elements over the xylem fibers, even though both are composed of secondary cell walls. Fluorescence correlation spectroscopy, employed to analyze the interaction of expansins with isolated xylem, indicates that binding is governed by more than one factor, which could include interaction with more than one type of polymer in the fibers, such as cellulose and hemicellulose or pectin. Binding to different polysaccharides could explain the observed reduction of cellulolytic and xylanolytic activities in the presence of expansin, possibly because of competition for the substrate. Our findings are relevant for the comprehensive understanding of the pathogenesis by P. carotovorum during xylem invasion, a process in which Exl1 might be involved.

This study reports the effects of exposing cells of the prototypical enteropathogenic Escherichia coli (EPEC) strain E2348/69 to static magnetic fields (SMF) of varying intensities to observe their capacity to autoaggregate and the effect on cell adherence. The results showed that bacteria exposure over the course of 5 min to an intensity of 53 mT reduced autoaggregation by 28%. However, with intensities of up to 100 mT with the same exposure time, bacteria autoaggregation was reduced by approximately 50%; and after 30 min at the same intensity, it was indistinguishable from that observed in a non-autoaggregative strain. Furthermore, it was observed that SMF treatment also modified the typical localized adherence pattern of EPEC E2348/69. The observed effects are not related to bacteria damage. The above was confirmed because, after a 107 mT SMF treatment over the course of 30 min, cell viability and membrane permeability were the same to that observed in untreated controls. The obtained results suggest that the SMF effect on the E2348/69 EPEC strain alters the expression of the bundle-forming pilus (BFP), due to the fact that the same strain without the EPEC adherence factor plasmid that encodes the BFP operon was unable to autoaggregate. Electron microscopic analyses revealed structural differences between cells exposed to SMF with respect to untreated controls. In conclusion, the SMF treatment of 107 mT for 30 min reduced EPEC E2348/69 autoaggregation and modified its adherence pattern, with both events likely being associated with changes in BFP expression. Bioelectromagnetics. 38:570-578, 2017. © 2017 Wiley Periodicals, Inc.

Consensus engineering has been used to design more stable variants using the most frequent amino acid at each site of a multiple sequence alignment; sometimes consensus engineering modifies function, but efforts have mainly been focused on studying stability. Here we constructed a consensus Rossmann domain for the Shikimate dehydrogenase enzyme; separately we decided to switch the cofactor specificity through rational design in the Escherichia coli Shikimate dehydrogenase enzyme and then analyzed the effect of consensus mutations on top of our design. We found that consensus mutations closest to the 2' adenine moiety increased the activity in our design. Consensus engineering has been shown to result in more stable proteins and our findings suggest it could also be used as a complementary tool for increasing or modifying enzyme activity during design.

Since the publication of a landmark article on the structure of EXLX1 from Bacillus subtilis in 2011, our knowledge of bacterial expansins has steadily increased and our view and understanding of these enigmatic proteins has advanced with relation to their structure, phylogenetic relationships and substrate interaction, although the molecular basis for their mechanism of action remains to be determined. Lignocellulosic material represents a source of fermentable sugars for the production of biofuels, and cell-wall degrading activities are essential to efficiently release such sugars from their polymeric structures. Because expansins from fungi and bacteria seem to be required to properly colonize or cause disease to plant tissues, and because they share some characteristics with their plant counterparts for loosening the cell wall they have been seen as a promising tool to overcome the recalcitrance of these materials. However, microbial expansins activity is at best, very low compared with plant expansins activity. This revision analyses recent work on bacterial expansins structure, function and biological role, emphasizing our need to focus on their mechanism of action as a means to design better strategies for their use, in both in the energy and agricultural industries.

BACKGROUND:The cAMP-dependent protein kinase regulatory network (PKA-RN) regulates metabolism, memory, learning, development, and response to stress. Previous models of this network considered the catalytic subunits (CS) as a single entity, overlooking their functional individualities. Furthermore, PKA-RN dynamics are often measured through cAMP levels in nutrient-depleted cells shortly after being fed with glucose, dismissing downstream physiological processes.RESULTS:Here we show that temperature stress, along with deletion of PKA-RN genes, significantly affected HSE-dependent gene expression and the dynamics of the PKA-RN in cells growing in exponential phase. Our genetic analysis revealed complex regulatory interactions between the CS that influenced the inhibition of Hsf1/Skn7 transcription factors. Accordingly, we found new roles in growth control and stress response for Hsf1/Skn7 when PKA activity was low (cdc25Δ cells). Experimental results were used to propose an interaction scheme for the PKA-RN and to build an extension of a classic synchronous discrete modeling framework. Our computational model reproduced the experimental data and predicted complex interactions between the CS and the existence of a repressor of Hsf1/Skn7 that is activated by the CS. Additional genetic analysis identified Ssa1 and Ssa2 chaperones as such repressors. Further modeling of the new data foresaw a third repressor of Hsf1/Skn7, active only in the absence of Tpk2. By averaging the network state over all its attractors, a good quantitative agreement between computational and experimental results was obtained, as the averages reflected more accurately the population measurements.CONCLUSIONS:The assumption of PKA being one molecular entity has hindered the study of a wide range of behaviors. Additionally, the dynamics of HSE-dependent gene expression cannot be simulated accurately by considering the activity of single PKA-RN components (i.e., cAMP, individual CS, Bcy1, etc.). We show that the differential roles of the CS are essential to understand the dynamics of the PKA-RN and its targets. Our systems level approach, which combined experimental results with theoretical modeling, unveils the relevance of the interaction scheme for the CS and offers quantitative predictions for several scenarios (WT vs. mutants in PKA-RN genes and growth at optimal temperature vs. heat shock).

ABSTRACT Expansins are a family of proteins with plant cell wall remodeling‐activity, which bind cell wall components through hydrophobic and electrostatic interactions. A shallow area on the surface of the protein serves as the polysaccharide binding site (PBS) and it is composed of conserved residues. However, electric charge differences on the opposite face of the PBS produce basic, neutral, or acidic proteins. An analysis of forty‐four bacterial expansins, homologues of BsEXLX1, revealed two main groups defined by: (a) the presence or absence of disulfide bonds; and (b) by the proteins isoelectric point (pI). We determined the location of the residues responsible for the pI on the structure of representative expansins. Our results suggest that the electric charge at the opposite site of the PBS may help in substrate differentiation among expansins from different species; in addition, electrostatic polarization between the front and the back of the molecule could affect expansin activity on cellulose. Proteins 2015; 83:215–223. © 2014 Wiley Periodicals, Inc.

Microbial expansins act on plant cell walls similarly to plant expansins, albeit their loosening activity levels are tenfold lesser compared to plant expansins. We report the characterization of an expansin-like gene from the plant pathogen Pectobacterium carotovorum, named exl1. PcExl1 is an acidic protein that binds cellulose (Avicel), and weakens filter paper. The acidic nature of PcExl1 confers different binding properties when compared to Bacillus subtilis BsEXLX1, which is a basic protein. PcExl1 binding to wheat cell wall increased when acidic components were depleted, reaching a similar level to the binding to Avicel, indicating that cellulose is the target of PcExl1.