Domain Shuffling in a Sensor Protein Contributed to the Evolution of Insect Pathogenicity in Plant-Beneficial Pseudomonas protegens

Pseudomonas protegens is a biocontrol rhizobacterium with a plant-beneficial and an insect pathogenic lifestyle, but it is not understood how the organism switches between the two states. Here, we focus on understanding the function and possible evolution of a molecular sensor that enables P. protegens to detect the insect environment and produce a potent insecticidal toxin specifically during insect infection but not on roots. By using quantitative single cell microscopy and mutant analysis, we provide evidence that the sensor histidine kinase FitF is a key regulator of insecticidal toxin production. Our experimental data and bioinformatic analyses indicate that FitF shares a sensing domain with DctB, a histidine kinase regulating carbon uptake in Proteobacteria. This suggested that FitF has acquired its specificity through domain shuffling from a common ancestor. We constructed a chimeric DctB-FitF protein and showed that it is indeed functional in regulating toxin expression in P. protegens. The shuffling event and subsequent adaptive modifications of the recruited sensor domain were critical for the microorganism to express its potent insect toxin in the observed host-specific manner. Inhibition of the FitF sensor during root colonization could explain the mechanism by which P. protegens differentiates between the plant and insect host. Our study establishes FitF of P. protegens as a prime model for molecular evolution of sensor proteins and bacterial pathogenicity.     

Phylogenetically closely related pseudomonads isolated from arthropods exhibit differential insect-killing abilities and genetic variations in insecticidal factors

Strains belonging to the Pseudomonas protegens and Pseudomonas chlororaphis species are able to control soilborne plant pathogens and to kill pest insects by producing virulence factors such as toxins, chitinases, antimicrobials or two-partner secretion systems. Most insecticidal Pseudomonas described so far were isolated from roots or soil. It is unknown whether these bacteria naturally occur in arthropods and how they interact with them. Therefore, we isolated P. protegens and P. chlororaphis from various healthy insects and myriapods, roots and soil collected in an agricultural field and a neighbouring grassland. The isolates were compared for insect killing, pathogen suppression and host colonization abilities. Our results indicate that neither the origin of isolation nor the phylogenetic position mirror the degree of insecticidal activity. Pseudomonas protegens strains appeared homogeneous regarding phylogeny, biocontrol and insecticidal capabilities, whereas P. chlororaphis strains were phylogenetically and phenotypically more heterogenous. A phenotypic and genomic analysis of five closely related P. chlororaphis isolates displaying varying levels of insecticidal activity revealed variations in genes encoding insecticidal factors that may account for the reduced insecticidal activity of certain isolates. Our findings point towards an adaption to insects within closely related pseudomonads and contribute to understand the ecology of insecticidal Pseudomonas.     

Pivotal role of O-antigenic polysaccharide display in the sensitivity against phage tail-like particles in environmental Pseudomonas kin competition

Environmental pseudomonads colonize various niches including insect and plant environments. When invading these environments, bacteria are confronted with the resident microbiota. To oppose with closely related strains, they rely on narrow-spectrum weaponry such as tailocins, i.e., phage tail-like particles. Little is known about the receptors for these tailocins especially among phylogenetically closely related species. Here, we studied the interaction between an R-tailocin from Pseudomonas protegens CHA0 and a targeted kin, Pseudomonas protegens Pf-5. Using genome-wide transposon insertion sequencing, we identified that lipopolysaccharides are involved in the sensitivity of Pf-5 towards the tailocin of CHA0. By generating Pf-5 lipopolysaccharide mutants and exposing them to extracted tailocin, we specified the two O-antigenic polysaccharides (O-PS) targeted by the tailocin. We affirmed the role of these O-PS through competition assays in vitro as well as in insects. Further, we demonstrate that O-PS are double-edge swords that are responsible for the sensitivity of P. protegens towards tailocins and phages produced by their kin, but shield bacteria from the immune system of the insect. Our results shed light on the trade-off that bacteria are confronted with, where specific O-PS decorations can both be of benefit or disadvantage depending on the host environment and its bacterial inhabitants.Subject terms: Bacteriophages, Environmental microbiology     

Patch size drives colonization by aquatic insects,with minor priority effects of a cohabitant

Patch size is one of the most important factors affecting the distribution and abundance of species, and recent research has shown that patch size is an important niche dimension affecting community structure in aquatic insects. Building on this result, we examined the impact of patch size in conjunction with presence of larval anurans on colonization by aquatic insects. Hyla chrysoscelis (Cope''s gray treefrog) larvae are abundant and early colonists in fishless lentic habitats, and these larvae can fill multiple ecological roles. By establishing larvae in mesocosms prior to colonization, we were able to assess whether H. chrysoscelis larvae have priority effects on aquatic insect assemblages. We conducted a series of three experiments in naturally colonized experimental landscapes to test whether (1) H. chrysoscelis larval density affects insect colonization, (2) variation in patch size affects insect colonization, and (3) the presence and larval density of H. chrysoscelis shift colonization of insects between patches of different size. Larval density independently had almost no effect on colonization, while patch size had species‐specific effects consistent with prior work. When larvae and patch size were tested in conjunction, patch size had numerous, often strong, species‐specific effects on colonization; larval density had effects largely limited to the assemblages of colonizing beetles and water bugs, with few effects on individual species. Higher larval densities in large mesocosms shifted some insect colonization to smaller patches, resulting in higher beta diversity among small patches in proximity to high density large mesocosms. This indicates establishing H. chrysoscelis larvae prior to insect colonization can likely create priority effects that slightly shape insect communities. Our results support the importance of patch size in studying species abundances and distributions and also indicate that colonization order plays an important role in determining the communities found within habitat patches.     

Colonization of eucalypts in New Zealand by Australian insects

Since the 1860s, Australian insects have steadily colonized eucalypts in New Zealand. The rate of colonization has increased markedly over the last two decades. This increase may be related to increasing trade between the two countries. Currently there are 26 specialist eucalypt insect species and approximately 31 polyphagous insect species that can feed on Eucalyptus in New Zealand. The specialist eucalypt insects endemic to Australia have generally caused more damage than polyphagous or native insects. Eucalypt‐specific insects are dominated by sap sucking bugs, particularly psyllids, and defoliating Coleoptera and Lepidoptera. In some cases the major insect pest species have been those that are only occasional pests in Australia, for example Gonipterus scutellatus, Ctenarytaina eucalypti, Eriococcus coriaceus and Phylacteophaga froggatti. Some important insect pests have been rare, or even not described from Australia, prior to their appearance as a pest in New Zealand, for example Paropsis charybdis and Ophelimus eucalypti. Invading eucalypt insects are more likely to establish in the Auckland region than anywhere else in New Zealand.     

Root-to-Root Travel of the Beneficial Bacterium Azospirillum brasilense

The root-to-root travel of the beneficial bacterium Azospirillum brasilense on wheat and soybean roots in agar, sand, and light-textured soil was monitored. We used a motile wild-type (Mot⁺) strain and a motility-deficient (Mot^-) strain which was derived from the wild-type strain. The colonization levels of inoculated roots were similar for the two strains. Mot⁺ cells moved from inoculated roots (either natural or artificial roots in agar, sand, or light-textured soil) to noninoculated roots, where they formed a band-type colonization composed of bacterial aggregates encircling a limited part of the root, regardless of the plant species. The Mot^- strain did not move toward noninoculated roots of either plant species and usually stayed at the inoculation site and root tips. The effect of attractants and repellents was the primary factor governing the motility of Mot⁺ cells in the presence of adequate water. We propose that interroot travel of A. brasilense is an essential preliminary step in the root-bacterium recognition mechanism. Bacterial motility might have a general role in getting Azospirillum cells to the site where firmer attachment favors colonization of the root system. Azospirillum travel toward plants is a nonspecific active process which is not directly dependent on nutrient deficiency but is a consequence of a nonspecific bacterial chemotaxis, influenced by the balance between attractants and possibly repellents leaked by the root.     

Stages of Infection during the Tripartite Interaction between Xenorhabdus nematophila, Its Nematode Vector, and Insect Hosts

Bacteria of the genus Xenorhabdus are mutually associated with entomopathogenic nematodes of the genus Steinernema and are pathogenic to a broad spectrum of insects. The nematodes act as vectors, transmitting the bacteria to insect larvae, which die within a few days of infection. We characterized the early stages of bacterial infection in the insects by constructing a constitutive green fluorescent protein (GFP)-labeled Xenorhabdus nematophila strain. We injected the GFP-labeled bacteria into insects and monitored infection. We found that the bacteria had an extracellular life cycle in the hemolymph and rapidly colonized the anterior midgut region in Spodoptera littoralis larvae. Electron microscopy showed that the bacteria occupied the extracellular matrix of connective tissues within the muscle layers of the Spodoptera midgut. We confirmed the existence of such a specific infection site in the natural route of infection by infesting Spodoptera littoralis larvae with nematodes harboring GFP-labeled Xenorhabdus. When the infective juvenile (IJ) nematodes reached the insect gut, the bacterial cells were rapidly released from the intestinal vesicle into the nematode intestine. Xenorhabdus began to escape from the anus of the nematodes when IJs were wedged in the insect intestinal wall toward the insect hemolymph. Following their release into the insect hemocoel, GFP-labeled bacteria were found only in the anterior midgut region and hemolymph of Spodoptera larvae. Comparative infection assays conducted with another insect, Locusta migratoria, also showed early bacterial colonization of connective tissues. This work shows that the extracellular matrix acts as a particular colonization site for X. nematophila within insects.     

Gene expression of serine and cysteine proteinase inhibitors during cereal leaf beetle larvae feeding on wheat: the role of insect-associated microorganisms

Plants and insects have been coexisting for more than 350 million years. During this time, both have evolved many strategies to successfully exploit or respond to reciprocal adaptation and defense reactions. Plants tend to minimize the damage caused by pest feeding, while pests tend to manipulate plant response by suppressing plant defense mechanisms or developing strategies to overcome plant defense systems. Plants recognize insect pests by the wounding that they cause and elicitors present in pest oral secretions (saliva and/or regurgitant). These elicitors or insect-associated microorganisms can modulate plant response to the benefit of their insect hosts. In this article, we have undertaken an analysis of gene expression in serine and cysteine proteinase inhibitors (SerPI and CysPI, respectively) in wheat (Triticum aestivum) plants exposed to cereal leaf beetle (CLB, Oulema melanopus, Coleoptera, Chrysomelidae) larvae feeding, and the impact of microbes associated with CLB on the expression levels of these genes. Using three wheat varieties and antibiotic-treated and untreated CLB larvae, we found that SerPI plays a more important role than CysPIs in plant defense against CLB larvae. Additionally, higher levels of SerPI gene expression were observed in systemic leaves in comparison to the wounded leaves (local response). Each of the tested wheat varieties reacted in a specific way to the particular treatment. Moreover, the presence of microbial components associated with insects influenced plant response to CLB larvae feeding.     

Site-Specific Cassette Exchange Systems in the Aedes aegypti Mosquito and the Plutella xylostella Moth

Genetically engineered insects are being evaluated as potential tools to decrease the economic and public health burden of mosquitoes and agricultural pest insects. Here we describe a new tool for the reliable and targeted genome manipulation of pest insects for research and field release using recombinase mediated cassette exchange (RMCE) mechanisms. We successfully demonstrated the established ΦC31-RMCE method in the yellow fever mosquito, Aedes aegypti, which is the first report of RMCE in mosquitoes. A new variant of this RMCE system, called iRMCE, combines the ΦC31-att integration system and Cre or FLP-mediated excision to remove extraneous sequences introduced as part of the site-specific integration process. Complete iRMCE was achieved in two important insect pests, Aedes aegypti and the diamondback moth, Plutella xylostella, demonstrating the transferability of the system across a wide phylogenetic range of insect pests.     

Expression of insect α6-like nicotinic acetylcholine receptors in Drosophila melanogaster highlights a high level of conservation of the receptor:spinosyn interaction

Insecticide research has often relied on model species for elucidating the resistance mechanisms present in the targeted pests. The accuracy and applicability of extrapolations of these laboratory findings to field conditions varies but, for target site resistance, conserved mechanisms are generally the rule rather than the exception (Perry et al., 2011). The spinosyn class of insecticides appear to fit this paradigm and are a pest control option with many uses in both crop and animal protection. Resistance to spinosyns has been identified in both laboratory-selected and field-collected pest insects.Studies using the model insect, Drosophila melanogaster, have identified the nicotinic acetylcholine receptor subunit, Dα6 as an important target of the insecticide spinosad (Perry et al., 2007, Watson et al., 2010). Field-isolated resistant strains of several agricultural pest insects provide evidence that resistance cases are often associated with mutations in orthologues to Dα6 (Baxter et al., 2010, Puinean et al., 2013).The expression of these receptors is difficult in heterologous systems. In order to examine the biology of the Dα6 receptor subunit further, we used Drosophila as a model and developed an in vivo rescue system. This allowed us to express four different isoforms of Dα6 and show that each is able to rescue the response to spinosad. Regulatory sequences upstream of the Dα6 gene able to rescue the resistance phenotype were identified. Expression of other D. melanogaster subunits revealed that the rescue phenotype appears to be Dα6 specific. We also demonstrate that expression of pest insect orthologues of Dα6 from a variety of species are capable of rescuing the spinosad response phenotype, verifying the relevance of this receptor to resistance monitoring in the field. In the absence of a robust heterologous expression system, this study presents an in vivo model that will be useful in analysing many other aspects of these receptors and their biology.     

Water stress and kaolin spray affect herbivorous insects’ success on cotton

Ecological hypotheses of plant–insect herbivore interactions suggest that insects perform better on weakened plants and plants grown under optimal conditions are less damaged. This study tested the hypothesis that the colonization and oviposition rates by pests with different feeding strategies and levels of specialization are affected in different ways by two conditions commonly faced by commercially grown plants–water deficit and application of kaolin sprays, a reducer of abiotic plant stressors. We used four major pests of cotton as insect herbivore models. Three were chewing Lepidoptera: Alabama argillacea (Hüb.), a monophagous pest on cotton; Heliothis virescens (Fabr.), which is polyphagous, but with cotton as a primary host; and Chrysodeixis includens (Walk.), which is polyphagous, with cotton as secondary host. The fourth pest was a sap-sucking species, the polyphagous whitefly Bemisia tabaci (Gen.). In both choice and no-choice trials, the three chewing pests oviposited significantly less upon water-stressed plants; the greatest effect was observed for C. includens (>90 % reduction in oviposition under choice and >58 % under no-choice conditions). In contrast, the sap-sucking B. tabaci exhibited statistically more colonization and oviposition on water-stressed plants. Application of kaolin sprays reduced colonization and oviposition by all herbivore species tested, irrespective of irrigation regime and feeding strategies.     

A Novel Interaction between Plant-Beneficial Rhizobacteria and Roots: Colonization Induces Corn Resistance against the Root Herbivore Diabrotica speciosa

A number of soil-borne microorganisms, such as mycorrhizal fungi and rhizobacteria, establish mutualistic interactions with plants, which can indirectly affect other organisms. Knowledge of the plant-mediated effects of mutualistic microorganisms is limited to aboveground insects, whereas there is little understanding of what role beneficial soil bacteria may play in plant defense against root herbivory. Here, we establish that colonization by the beneficial rhizobacterium Azospirillum brasilense affects the host selection and performance of the insect Diabrotica speciosa. Root larvae preferentially orient toward the roots of non-inoculated plants versus inoculated roots and gain less weight when feeding on inoculated plants. As inoculation by A. brasilense induces higher emissions of (E)-β-caryophyllene compared with non-inoculated plants, it is plausible that the non-preference of D. speciosa for inoculated plants is related to this sesquiterpene, which is well known to mediate belowground insect-plant interactions. To the best of our knowledge, this is the first study showing that a beneficial rhizobacterium inoculant indirectly alters belowground plant-insect interactions. The role of A. brasilense as part of an integrative pest management (IPM) program for the protection of corn against the South American corn rootworm, D. speciosa, is considered.     

Plant tissue localization of the endophytic insect pathogenic fungi Metarhizium and Beauveria

Endophytic fungi may display preferential tissue colonization within their plant hosts. Here we tested if the endophytic, insect pathogenic fungi (EIPF) Metarhizium and Beauveria showed preferential localization within plant tissues, in the field and under laboratory conditions. In the field, plants were sampled from three separate sites (Brock University, St. Catharines, Ontario; Pelham, Ontario; and Torngat Mountains National Park, Newfoundland, Canada) and EIPF were isolated from plant roots, the hypocotyl, and stem and leaves. Two genera of EIPF, Metarhizium spp. and Beauveria bassiana, were isolated from plants sampled, as well as the nematophagous fungus, Pochonia chlamydosporium. Metarhizium spp. were almost exclusively found in roots, whereas B. bassiana and P. chlamydosporium were found throughout the plant. The Metarhizium species were identified by RFLP and 95 % were Metarhizium robertsii, 4.3 % were M. brunneum, and 0.7 % were M. guizhouense. Lab studies with M. robertsii and B. bassiana reflected observations found in the field, that is, Metarhizium was restricted to the roots of plants while B. bassiana was found throughout the plant. Insect infection by these EIPF is preferential with respect to above and below ground insects, and the present study correlates above and below ground insect infections with endophytic colonization by these EIPF.     

Lack of Host Specialization in Aspergillus flavus

Aspergillus spp. cause disease in a broad range of organisms, but it is unknown if strains are specialized for particular hosts. We evaluated isolates of Aspergillus flavus, Aspergillus fumigatus, and Aspergillus nidulans for their ability to infect bean leaves, corn kernels, and insects (Galleria mellonella). Strains of A. flavus did not affect nonwounded bean leaves, corn kernels, or insects at 22°C, but they killed insects following hemocoelic challenge and caused symptoms ranging from moderate to severe in corn kernels and bean leaves injured during inoculation. The pectinase P2c, implicated in aggressive colonization of cotton bolls, is produced by most A. flavus isolates, but its absence did not prevent colonization of bean leaves. Proteases have been implicated in colonization of animal hosts. All A. flavus strains produced very similar patterns of protease isozymes when cultured on horse lung polymers. Quantitative differences in protease levels did not correlate with the ability to colonize insects. In contrast to A. flavus, strains of A. nidulans and A. fumigatus could not invade living insect or plant tissues or resist digestion by insect hemocytes. Our results indicate that A. flavus has parasitic attributes that are lacking in A. fumigatus and A. nidulans but that individual strains of A. flavus are not specialized to particular hosts.     

A New Approach to Quantify Semiochemical Effects on Insects Based on Energy Landscapes

Introduction

Our ability to document insect preference for semiochemicals is pivotal in pest control as these agents can improve monitoring and be deployed within integrated pest management programmes for more efficacious control of pest species. However, methods used to date have drawbacks that limit their utility. We present and test a new concept for determining insect motivation to move towards, or away from, semiochemicals by noting direction and speed of movement as animals work against a defined energy landscape (environmentally dependent variation in the cost of transport) requiring different powers to negotiate. We conducted trials with the pine weevils Hylobius abietis and peach-potato aphids Myzus persicae exposed to various attractants and repellents and placed so that they either moved up defined slopes against gravity or had to travel over variously rough surfaces.

Results

Linear Mixed Models demonstrated clear reductions in travel speed by insects moving along increasingly energetically taxing energy landscapes but also that responses varied according to different semiochemicals, thus highlighting the value of energy landscapes as a new concept to help measure insect motivation to access or avoid different attractants or repellents across individuals.

Conclusions

New sensitive, detailed indicators of insect motivation derived from this approach should prove important in pest control across the world.     

Metarhizium robertsii Produces an Extracellular Invertase (MrINV) That Plays a Pivotal Role in Rhizospheric Interactions and Root Colonization

As well as killing pest insects, the rhizosphere competent insect-pathogenic fungus Metarhizium robertsii also boosts plant growth by providing nitrogenous nutrients and increasing resistance to plant pathogens. Plant roots secrete abundant nutrients but little is known about their utilization by Metarhizium spp. and the mechanistic basis of Metarhizium-plant associations. We report here that M. robertsii produces an extracellular invertase (MrInv) on plant roots. Deletion of MrInv (⊿MrInv) reduced M. robertsii growth on sucrose and rhizospheric exudates but increased colonization of Panicum virgatum and Arabidopsis thaliana roots. This could be accounted for by a reduction in carbon catabolite repression in ⊿MrInv increasing production of plant cell wall-degrading depolymerases. A non-rhizosphere competent scarab beetle specialist Metarhizium majus lacks invertase which suggests that rhizospheric competence may be related to the sugar metabolism of different Metarhizium species.     

Functional analysis of a novel orthologous small heat shock protein (shsp) hsp21.8a and seven species-specific shsps in Tribolium castaneum

Small heat shock proteins (sHSPs) are important modulators of insect survival. Previous research revealed that there is only one orthologous cluster of shsps in insects. Here, we identified another novel orthologous cluster of shsps in insects by comparative analysis. Multiple stress experiments and function investigation of Tchsp21.8a belonging to this orthologous cluster and seven species-specific shsps were performed in the stored-grain pest Tribolium castaneum. The results indicated that expression of Tchsp21.8a showed weak responses to different stresses. However, expressions of most species-specific shsps exhibited hyper-responses to heat stress, and expressions of all species-specific shsps displayed diverse responses during other stresses to protect beetles in a cooperative manner. Additionally, Tchsp21.8a and species-specific Tcshsp19.7 played important roles in the development of T. castaneum, and all Tcshsps had a certain impact on the fecundity. Our work created a comprehensive reliable scaffold of insect shsps that can further provide instructive insights to pest bio-control     

Oosporein,an abundant metabolite in Beauveria caledonica,with a feedback induction mechanism and a role in insect virulence

Oosporein was first identified from the insect pathogen Beauveria bassiana >50 y ago. Here, we investigate the insecticidal, anti-feedant and immunomodulation effects of oosporein produced by Beauveria caledonica on the forestry pest Hylobius abietis and model insect Galleria mellonella. We report a novel feedback induction mechanism regulating oosporein production in B. caledonica; exogenous oosporein induces the expression of the oosporein cluster, leading to increased abundance of oosporein biosynthetic enzymes, as shown by label-free quantitative proteomics. Oosporein did not have an anti-feedant effect on H. abietis adults – on the contrary, insects exposed to oosporein-treated food fed more than those exposed to untreated food only. Injected oosporein did not kill insect larvae but increased susceptibility of H. abietis to a subsequent infection. Oosporein did not act as a contact toxin on H. abietis adults and G. mellonella larvae at the concentrations tested. Therefore, it appears that oosporein promotes infection rather than directly killing insects; this could be mediated both by a reduction in haemocyte numbers and by alterations to the humoral immune system. This work makes a case for future research into the potential use of B. caledonica as a biocontrol agent through combinations with oosporein or with enhanced production of oosporein.     

Constitutive Activation of the Midgut Response to Bacillus thuringiensis in Bt-Resistant Spodoptera exigua

Bacillus thuringiensis is the most effective microbial control agent for controlling numerous species from different insect orders. The main threat for the long term use of B. thuringiensis in pest control is the ability of insects to develop resistance. Thus, the identification of insect genes involved in conferring resistance is of paramount importance. A colony of Spodoptera exigua (Lepidoptera: Noctuidae) was selected for 15 years in the laboratory for resistance to Xentari™, a B. thuringiensis-based insecticide, reaching a final resistance level of greater than 1,000-fold. Around 600 midgut ESTs were analyzed by DNA-macroarray in order to find differences in midgut gene expression between susceptible and resistant insects. Among the differentially expressed genes, repat and arylphorin were identified and their increased expression was correlated with B. thuringiensis resistance. We also found overlap among genes that were constitutively over-expressed in resistant insects with genes that were up-regulated in susceptible insects after exposure to Xentari™, suggesting a permanent activation of the response to Xentari™ in resistant insects. Increased aminopeptidase activity in the lumen of resistant insects in the absence of exposure to Xentari™ corroborated the hypothesis of permanent activation of response genes. Increase in midgut proliferation has been proposed as a mechanism of response to pathogens in the adult from several insect species. Analysis of S. exigua larvae revealed that midgut proliferation was neither increased in resistant insects nor induced by exposure of susceptible larvae to Xentari™, suggesting that mechanisms other than midgut proliferation are involved in the response to B. thuringiensis by S. exigua larvae.