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Ralstonia pseudosolanacearum, a notorious phytopathogen, is responsible for causing bacterial wilt, leading to significant economic losses globally in many crops within the Solanaceae family. Despite various cultural and chemical control strategies, managing bacterial wilt remains a substantial challenge. This study demonstrates, for the first time, the effective use of plant-induced bacterial gene silencing against R. pseudosolanacearum, facilitated by Tobacco rattle virus-mediated gene silencing, to control bacterial wilt symptoms in Nicotiana benthamiana. The methodology described in this study could be utilized to identify novel phytobacterial virulence factors through both forward and reverse genetic approaches. To validate plant-induced gene silencing, small RNA fractions extracted from plant exudates were employed to silence bacterial gene expression, as indicated by the reduction in the expression of GFP and virulence genes in R. pseudosolanacearum. Furthermore, treatment of human and plant pathogenic Gram-negative and Gram-positive bacteria with plant-generated small RNAs resulted in the silencing of target genes within 48 hours. Taken together, the results suggest that this technology could be applied under field conditions, offering precise, gene-based control of target bacterial pathogens while preserving the indigenous microbiota.
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The spatial organization of gut microbiota is crucial for the functioning of the gut ecosystem, although the mechanisms that organize gut bacterial communities in microhabitats are only partially understood. The gut of the insect Riptortus pedestris has a characteristic microbiota biogeography with a multispecies community in the anterior midgut and a monospecific bacterial population in the posterior midgut. We show that the posterior midgut region produces massively hundreds of specific antimicrobial peptides (AMPs), the Crypt-specific Cysteine-Rich peptides (CCRs) that have membrane-damaging antimicrobial activity against diverse bacteria but posterior midgut symbionts have elevated resistance. We determined by transposon-sequencing the genetic repertoire in the symbiont Caballeronia insecticola to manage CCR stress, identifying different independent pathways, including AMP-resistance pathways unrelated to known membrane homeostasis functions as well as cell envelope functions. Mutants in the corresponding genes have reduced capacity to colonize the posterior midgut, demonstrating that CCRs create a selective barrier and resistance is crucial in gut symbionts. Moreover, once established in the gut, the bacteria differentiate into a CCR-sensitive state, suggesting a second function of the CCR peptide arsenal in protecting the gut epithelia or mediating metabolic exchanges between the host and the gut symbionts. Our study highlights the evolution of an extreme diverse AMP family that likely contributes to establish and control the gut microbiota.
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Péptidos Antimicrobianos , Microbioma Gastrointestinal , Simbiosis , Animales , Péptidos Antimicrobianos/metabolismo , Péptidos Antimicrobianos/genética , Péptidos Antimicrobianos/farmacología , Bacterias/genética , Bacterias/metabolismo , Bacterias/efectos de los fármacos , Tracto Gastrointestinal/microbiologíaRESUMEN
Insects lack acquired immunity and were thought to have no immune memory, but recent studies reported a phenomenon called immune priming, wherein sublethal dose of pathogens or nonpathogenic microbes stimulates immunity and prevents subsequential pathogen infection. Although the evidence for insect immune priming is accumulating, the underlying mechanisms are still unclear. The bean bug Riptortus pedestris acquires its gut microbiota from ambient soil and spatially structures them into a multispecies and variable community in the anterior midgut and a specific, monospecies Caballeronia symbiont population in the posterior region. We demonstrate that a particular Burkholderia strain colonizing the anterior midgut stimulates systemic immunity by penetrating gut epithelia and migrating into the hemolymph. The activated immunity, consisting of a humoral and a cellular response, had no negative effect on the host fitness, but on the contrary protected the insect from subsequent infection by pathogenic bacteria. Interruption of contact between the Burkholderia strain and epithelia of the gut weakened the host immunity back to preinfection levels and made the insects more vulnerable to microbial infection, demonstrating that persistent acquisition of environmental bacteria is important to maintain an efficient immunity.
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Burkholderia , Burkholderiaceae , Animales , Endodermo , Insectos , SueloRESUMEN
In recent years, the idea has flourished that plants emit and perceive sound and could even be capable of exchanging information through the acoustic channel. While research into plant bioacoustics is still in its infancy, with potentially fascinating discoveries awaiting ahead, here we show that the current knowledge is not conclusive. While plants do emit sounds under biotic and abiotic stresses such as drought, these sounds are high-pitched, of low intensity, and propagate only to a short distance. Most studies suggesting plant sensitivity to airborne sound actually concern the perception of substrate vibrations from the soil or plant part. In short, while low-frequency, high-intensity sounds emitted by a loudspeaker close to the plant seem to have tangible effects on various plant processes such as growth - a finding with possible applications in agriculture - it is unlikely that plants can perceive the sounds they produce, at least over long distances. So far, there is no evidence of plants communicating with each other via the acoustic channel.
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Acústica , Plantas , Sonido , Fenómenos Fisiológicos de las PlantasRESUMEN
Bacillus velezensis strain GB03 is a Gram-positive rhizosphere bacterium known for its ability to promote plant growth and immunity. This review provides a comprehensive overview of the research on GB03 from its initial discovery in Australian wheat fields in 1971 to its current applications. Recognized as a model plant growth-promoting rhizobacterium (PGPR), GB03 has exhibited outstanding performance in enhancing the growth and protection of many crop plants including cucumber, pepper, wheat, barley, soybean, and cotton. Notably, GB03 has been reported to elicit plant immune response, referred to as induced systemic resistance (ISR), against above-ground pathogens and insect pests. Moreover, a pivotal finding in GB03 was the first-ever identification of its bacterial volatile compounds, which are known to boost plant growth and activate ISR. Research conducted over the past five decades has clearly demonstrated the potential of GB03 as an eco-friendly substitute for conventional pesticides and fertilizers. Validating its safety, the U.S. Environmental Protection Agency endorsed GB03 for commercial use as Kodiak® in 1998. Subsequently, other compounds, such as BioYield™, were released as a biological control agent against soil-borne pathogens and as a biofertilizer, utilizing a durable spore formulation. More recently, GB03 has been utilized as a keystone modulator for engineering the rhizosphere microbiome and for eliciting microbe-induced plant volatiles. These extensive studies on GB03 underscore its significant role in sustainable agriculture, positioning it as a safe and environmentally-friendly solution for crop protection.
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A Gram-stain-negative, aerobic, reddish-coloured, rod-shaped and non-motile strain PAMC 29467T, was isolated from freshwater of the pond in Cambridge Bay, Canada. Strain PAMC 29467T was closely related to Hymenobacter yonginensis (98.1â% 16S rRNA gene similarity). Genomic relatedness analyses showed that strain PAMC 29467T is distinguishable from H. yonginensis based on average nucleotide identity (91.3â%) and digital DNA-DNA hybridization values (39.3â%). The major fatty acids (>10â%) of strain PAMC 29467T were summed feature 3 (C16â:â1 ω7c and/or C16â:â1 ω6c), C15â:â0 iso, C16â:â1 ω5c and summed feature 4 (C17â:â1 iso l and/or anteiso B). The major respiratory quinone was menaquinone-7. The genomic DNA G+C content was 61.5âmol%. Strain PAMC 29467T was separated from the type species in the genus Hymenobacter by its distinct phylogenetic position and some physiological characteristics. As a result, a novel species is proposed, with the name Hymenobacter canadensis sp. nov. (type strain, PAMC 29467T=KCTC 92787T=JCM 35843T).
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Cytophagaceae , Ácidos Grasos , Ácidos Grasos/química , Estanques , Filogenia , ARN Ribosómico 16S/genética , Bahías , Análisis de Secuencia de ADN , ADN Bacteriano/genética , Composición de Base , Técnicas de Tipificación Bacteriana , Agua Dulce , Vitamina K 2RESUMEN
Many insects possess symbiotic bacteria in their bodies, and microbial symbionts play pivotal metabolic roles for their hosts. Members of the heteropteran superfamilies Coreoidea and Lygaeoidea stinkbugs harbor symbionts of the genus Caballeronia in their intestinal tracts. Compared with symbiotic associations in Coreoidea, those in Lygaeoidea insects are still less understood. Here, we investigated a symbiotic relationship involving the mulberry seed bug Paradieuches dissimilis (Lygaeoidea: Rhyparochromidae) using histological observations, cultivation of the symbiont, 16S rRNA gene amplicon sequencing, and infection testing of cultured symbionts. Histological observations and cultivation revealed that P. dissimilis harbors Caballeronia symbionts in the crypts of its posterior midgut. 16S rRNA gene amplicon sequencing of field-collected P. dissimilis confirmed that the genus Caballeronia is dominant in the midgut of natural populations of P. dissimilis. In addition, PCR diagnostics showed that the eggs were free of symbiotic bacteria, and hatchlings horizontally acquired the symbionts from ambient soil. Infection and rearing experiments revealed that symbiont-free aposymbiotic individuals had abnormal body color, small body size, and, strikingly, a low survival rate, wherein no individuals reached adulthood, indicating an obligate cooperative mutualism between the mulberry seed bug and Caballeronia symbionts.
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Burkholderiaceae , Heterópteros , Morus , Humanos , Animales , Adulto , Simbiosis , ARN Ribosómico 16S/genética , Filogenia , Heterópteros/genética , Heterópteros/microbiología , Insectos , BacteriasRESUMEN
A Gram-stain-negative, aerobic, orange-coloured, rod-shaped and non-motile bacterial strain, PAMC 29362T, was isolated from an Antarctic lichen, Megaspora verrucosa. Phylogenetic and phylogenomic analyses indicated that strain PAMC 29362T belongs to the genus Polymorphobacter and was most closely related to Polymorphobacter arshaanensis (97.0% of 16S rRNA gene similarity), Polymorphobacter fuscus (96.3â%), Polymorphobacter multimanifer (95.3â%) and Polymorphobacter glacialis (95.2â%). Genomic relatedness analyses showed that strain PAMC 29362T is clearly distinguished from type strains of the genus Polymorphobacter based on values of average nucleotide identity (<74.3â%) and digital DNA-DNA hybridization (<20.4â%). The genomic DNA G+C content of PAMC 29362T was 65.5â%. The major fatty acids (>10â%) were summed feature 8 (C18:1 ω7c; 38.5â%) and summed feature 3 (C16:1 ω7c and/or C16:1 ω6c; 31.5â%). The major respiratory quinone was Q-10. Based on the results of phylogenetic, genome-based relatedness and physiological analyses, strain PAMC 29362T is proposed to represent a novel species of the genus Polymorphobacter, with the name Polymorphobacter megasporae sp. nov. The type strain is PAMC 29362T (=KCTC 82â578T=JCM 34545T).
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Alphaproteobacteria , Líquenes , Alphaproteobacteria/genética , Técnicas de Tipificación Bacteriana , Composición de Base , ADN Bacteriano/genética , Ácidos Grasos/química , Nucleótidos , Fosfolípidos , Filogenia , Quinonas , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
The bean bug Riptortus pedestris obtains a specific bacterial symbiont, Caballeronia insecticola (Burkholderia insecticola), from the environmental soil and harbors it in the posterior midgut region that is composed of hundreds of crypts. While newly hatched aposymbiotic insects possess primordial midgut crypts with little or no lumen, colonization of C. insecticola triggers swift development of the symbiotic organ, forming enlarged and opened crypts, and the symbiont subsequently fills the luminal cavities of those mature crypts. The cellular processes of crypt development triggered by C. insecticola colonization are poorly understood. Here we identified a fundamental mechanism of the symbiont-mediated midgut development by investigating cell cycles of intestinal epithelial cells. Intestinal stem cells of the bean bug are located and proliferate at the crypt base. Differentiated enterocytes migrate upward along the epithelial cell layer of the crypt as the midgut develops, induction of apoptosis in enterocytes primarily occurred on the tip side of the crypts, and apoptotic cells then eventually were shed from the crypts into the hemolymph. The proliferation rate of the stem cells at the base of the crypts was low while a high apoptotic rate was observed at the crypt tip in aposymbiotic insects, resulting in undeveloped short crypts. On the contrary, the gut-colonizing C. insecticola promoted the proliferation of the stem cells at the base of crypts and simultaneously inhibited apoptosis at the tip of crypts, resulting in a net growth of the crypts and the generation of a crypt lumen that becomes colonized by the bacterial symbiont. These results demonstrated that the Caballeronia symbiont colonization induces the development of the midgut crypts via finely regulating the enterocyte cell cycles, enabling it to stably and abundantly colonize the generated spacious crypts of the bean bug host.
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Insecticide resistance is one of the most serious problems in contemporary agriculture and public health. Although recent studies revealed that insect gut symbionts contribute to resistance, the symbiont-mediated detoxification process remains unclear. Here we report the in vivo detoxification process of an organophosphorus insecticide, fenitrothion, in the bean bug Riptortus pedestris. Using transcriptomics and reverse genetics, we reveal that gut symbiotic bacteria degrade this insecticide through a horizontally acquired insecticide-degrading enzyme into the non-insecticidal but bactericidal compound 3-methyl-4-nitrophenol, which is subsequently excreted by the host insect. This integrated "host-symbiont reciprocal detoxification relay" enables the simultaneous maintenance of symbiosis and efficient insecticide degradation. We also find that the symbiont-mediated detoxification process is analogous to the insect genome-encoded fenitrothion detoxification system present in other insects. Our findings highlight the capacity of symbiosis, combined with horizontal gene transfer in the environment, as a powerful strategy for an insect to instantly eliminate a toxic chemical compound, which could play a critical role in the human-pest arms race.
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Insecticidas/farmacología , Animales , Burkholderia/efectos de los fármacos , Burkholderia/genética , Heterópteros/efectos de los fármacos , Heterópteros/genética , Resistencia a los Insecticidas , Compuestos Organofosforados/farmacología , Simbiosis/efectos de los fármacos , Simbiosis/genéticaRESUMEN
Most animals harbor a gut microbiota that consists of potentially pathogenic, commensal, and mutualistic microorganisms. Dual oxidase (Duox) is a well described enzyme involved in gut mucosal immunity by the production of reactive oxygen species (ROS) that antagonizes pathogenic bacteria and maintains gut homeostasis in insects. However, despite its nonspecific harmful activity on microorganisms, little is known about the role of Duox in the maintenance of mutualistic gut symbionts. Here we show that, in the bean bug Riptortus pedestris, Duox-dependent ROS did not directly contribute to epithelial immunity in the midgut in response to its mutualistic gut symbiont, Burkholderia insecticola Instead, we found that the expression of Duox is tracheae-specific and its down-regulation by RNAi results in the loss of dityrosine cross-links in the tracheal protein matrix and a collapse of the respiratory system. We further demonstrated that the establishment of symbiosis is a strong oxygen sink triggering the formation of an extensive network of tracheae enveloping the midgut symbiotic organ as well as other organs, and that tracheal breakdown by Duox RNAi provokes a disruption of the gut symbiosis. Down-regulation of the hypoxia-responsive transcription factor Sima or the regulators of tracheae formation Trachealess and Branchless produces similar phenotypes. Thus, in addition to known roles in immunity and in the formation of dityrosine networks in diverse extracellular matrices, Duox is also a crucial enzyme for tracheal integrity, which is crucial to sustain mutualistic symbionts and gut homeostasis. We expect that this is a conserved function in insects.
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Burkholderia/crecimiento & desarrollo , Oxidasas Duales/metabolismo , Heterópteros , Proteínas de Insectos/metabolismo , Intestinos , Simbiosis/fisiología , Animales , Oxidasas Duales/genética , Heterópteros/enzimología , Heterópteros/genética , Heterópteros/microbiología , Proteínas de Insectos/genética , Intestinos/enzimología , Intestinos/microbiologíaRESUMEN
Resistance to toxins in insects is generally thought of as their own genetic trait, but recent studies have revealed that gut microorganisms could mediate resistance by detoxifying phytotoxins and man-made insecticides. By laboratory experiments, we here discovered a striking example of gut symbiont-mediated insecticide resistance in a serious rice pest, Cletus punctiger. The rice bug horizontally acquired fenitrothion-degrading Burkholderia through oral infection and housed it in midgut crypts. Fenitrothion-degradation test revealed that the gut-colonizing Burkholderia retains a high degrading activity of the organophosphate compound in the insect gut. This gut symbiosis remarkably increased resistance against fenitrothion treatment in the host rice bug. Considering that many stinkbug pests are associated with soil-derived Burkholderia, our finding strongly supports that a number of stinkbug species could gain resistance against insecticide simply by acquiring insecticide-degrading gut bacteria.
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Microbioma Gastrointestinal , Heterópteros , Oryza , Animales , Humanos , Resistencia a los Insecticidas , Laboratorios , SimbiosisRESUMEN
Insects harbor diverse microorganisms in the gut, providing their host with physiological and ecological advantages. For example, gut symbionts contribute to detoxification in phytophagous insects, degradation of lignocellulose in xylophagous insects, and in many insects, protection from pathogens by producing antimicrobial compounds. Furthermore, an unexpected function of the insect gut microbiota has been discovered-plastic degradation. Based on these diverse abilities of the insect gut microbiota that have been sophisticated under the natural environment, recent studies have aimed at applying the potential of gut microbes in the medical, engineering, and industrial fields.
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Bacterias/metabolismo , Microbioma Gastrointestinal/fisiología , Insectos/microbiología , Animales , Evolución Biológica , Digestión/fisiología , Ecosistema , Insectos/fisiología , Lignina/metabolismo , Plásticos/metabolismo , Proteínas Citotóxicas Formadoras de Poros/biosíntesisRESUMEN
In addition to abiotic triggers, biotic factors such as microbial symbionts can alter development of multicellular organisms. Symbiont-mediated morphogenesis is well-investigated in plants and marine invertebrates but rarely in insects despite the enormous diversity of insect-microbe symbioses. The bean bug Riptortus pedestris is associated with Burkholderia insecticola which are acquired from the environmental soil and housed in midgut crypts. To sort symbionts from soil microbiota, the bean bug develops a specific organ called the "constricted region" (CR), a narrow and symbiont-selective channel, located in the midgut immediately upstream of the crypt-bearing region. In this study, inoculation of fluorescent protein-labeled symbionts followed by spatiotemporal microscopic observations revealed that after the initial passage of symbionts through the CR, it closes within 12-18 h, blocking any potential subsequent infection events. The "midgut closure" developmental response was irreversible, even after symbiont removal from the crypts by antibiotics. It never occurred in aposymbiotic insects, nor in insects infected with nonsymbiotic bacteria or B. insecticola mutants unable to cross the CR. However, species of the genus Burkholderia and its outgroup Pandoraea that can pass the CR and partially colonize the midgut crypts induce the morphological alteration, suggesting that the molecular trigger signaling the midgut closure is conserved in this bacterial lineage. We propose that this drastic and quick alteration of the midgut morphology in response to symbiont infection is a mechanism for stabilizing the insect-microbe gut symbiosis and contributes to host-symbiont specificity in a symbiosis without vertical transmission.
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Infecciones Bacterianas , Burkholderia , Heterópteros , Animales , Burkholderia/genética , Sistema Digestivo , SimbiosisRESUMEN
Burkholderia sp. strain THE68 is a bacterial symbiont isolated from the midgut crypts of a phytophagous stink bug, Togo hemipterus Here, we report the complete 7.98-Mb genome of this symbiont, which consists of six circular replicons containing 7,238 protein coding genes.
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Despite the omnipresence of specific host-symbiont associations with acquisition of the microbial symbiont from the environment, little is known about how the specificity of the interaction evolved and is maintained. The bean bug Riptortus pedestris acquires a specific bacterial symbiont of the genus Burkholderia from environmental soil and harbors it in midgut crypts. The genus Burkholderia consists of over 100 species, showing ecologically diverse lifestyles, and including serious human pathogens, plant pathogens, and nodule-forming plant mutualists, as well as insect mutualists. Through infection tests of 34 Burkholderia species and 18 taxonomically diverse bacterial species, we demonstrate here that nonsymbiotic Burkholderia and even its outgroup Pandoraea could stably colonize the gut symbiotic organ and provide beneficial effects to the bean bug when inoculated on aposymbiotic hosts. However, coinoculation revealed that the native symbiont always outcompeted the nonnative bacteria inside the gut symbiotic organ, explaining the predominance of the native Burkholderia symbiont in natural bean bug populations. Hence, the abilities for colonization and cooperation, usually thought of as specific traits of mutualists, are not unique to the native Burkholderia symbiont but, to the contrary, competitiveness inside the gut is a derived trait of the native symbiont lineage only and was thus critical in the evolution of the insect gut symbiont.