Xenorhabdus bacteria, as pesticidal symbionts of the entomopathogenic nematodes Steinernema species, can contribute to this solution with a treasure trove of insecticidal compounds and an ability to suppress a variety of plant pathogens. As many challenges face sound exploitation of plant–phytonematode interactions, a full useful spectrum of such interactions should address nematicidal activity of Xenorhabdus. Steinernema–Xenorhabdus complex or Xenorhabdus individually should be involved in mechanisms underlying the favorable side of plant–nematode interactions in emerging cropping systems. Using Xenorhabdus bacteria should earnestly be harnessed to control not only phytonematodes, but also other plant pests and pathogens within integrated pest management plans. This review highlights the significance of fitting Xenorhabdus-obtained insecticidal, nematicidal, fungicidal, acaricidal, pharmaceutical, antimicrobial, and toxic compounds into existing, or arising, holistic strategies, for controlling many pests/pathogens. The widespread utilization of Xenorhabdus bacteria, however, has been slow-going, due to costs and some issues with their commercial processing. Yet, advances have been ongoing via further mastering of genome sequencing, discovering more of the beneficial Xenorhabdus species/strains, and their successful experimentations for pest control. Their documented pathogenicity to a broad range of arthropods and pathogens and versatility bode well for useful industrial products. The numerous beneficial traits of Xenorhabdus bacteria can facilitate their integration with other tactics for better pest/disease management programs.Mounting concern over the misuse of chemical pesticides has sparked broad interest for safe and effective alternatives to control plant pests and pathogens.  Xenorhabdus  individually or Steinernema (Steinernematidae: Rhabditida)–Xenorhabdus complex in emerging cropping systems for integrated pest management. These latter should be exploited not only for crop pest/pathogen management but also for sustainable agriculture.Upgrading the methods and materials used to control plant pests and pathogens via more effective and safer approaches is one of the current challenges in the field of agriculture. Growing discontent with many chemical pesticides has been elevating, due to their unacceptable impacts on human health and environmental contamination, non-target organisms, and plant-resistance development ,2,3. TheXenorhabdus comprises entomopathogenic bacterial species that can be promising alternatives for boosting the biocontrol of numerous plant pests/pathogens. These bacteria can secrete diverse arrays of operative bioactive metabolites against these pests [Xenorhabdus genes that encode for generating related compounds such as antibiotics, enzymes, bacteriocins, and toxins. They are established in the shape of pathogenicity islands on the chromosomes of different Xenorhabdus species.Admittedly, the significance of utilizing beneficial bacteria in various programs for pest/pathogen control has been increasing. While these bacteria can substitute the toxic and frequently undesirable chemicals employed for plant protection, they can offer benign ecological technologies . Among tse pests ,6,7,8,9.Xenorhabdus, with presupposed further genes encoding for beneficial attributes that are being detected in addition to presenting genes in the Xenorhabdus species that are already described so that it should be further exploited or their expressed produced compounds be improved [Xenorhabdus spp., it focuses on their up-to-date biotechnology to unlock more approaches of discovery that function for boosting durable agricultural systems. This review deals with the latest discoveries on their identification, taxonomy, diversity, and utilization in the present or future management plans, especially against plant pests/pathogens.Therefore, the current review addresses the diversity of endosymbiotic bacterial species from EPNs and their function in biological control against many plant pests and pathogens for the agro-environmental sustainability. It throws light on the recent discoveries of new species of improved ,9,10,11.Xenorhabdus and Photorhabdus bacteria  are phylogenetically close, it is not surprising that at first they were of the same genus [Xenorhabdus nematophila (type species) and X. luminesces) including symbionts of the nematode genera Steinernema and Heterorhabditis, respectively, were exclusively present until 1993 [X. nematophila from X. luminesces, leading to the transfer of all symbionts related to Heterorhabditis into Photorhabdus as a new genus with the type species Photorhabdus luminescens [Xenorhabdus and Photorhabdus bacteria as two recognized groups; P. luminescens distinctly had a DNA relatedness group unlike all Xenorhabdus strains with significant variations in phenotypic traits [Xenorhabdus bacteria are obviously separated from Photorhabdus species/strains by the 16S rDNA signature sequences [Photorhabdus luminescens and X. nematophila received much research work, due to being type species of their two genera, with high insecticidal activities and a global distribution.As me genus . Thus, ttil 1993 . Yet, thinescens . Before c traits . Xenorhaequences . Yet, boXenorhabdus still has more homogenous species than Photorhabdus, but the former genus possesses a higher number of species than the latter. For example, Sajnaga and Kazimierczak [Xenorhabdus spp. versus 19 Photorhabdus spp. This is possibly due to higher number of the current Steinernema species  than Heterorhabditis spp. , i.e., >100 Steinernema spp. but >20 Heterorhabditis spp. [Xenorhabdus and their Steinernema partner which are recognized or are likely to become recognized soon. In this respect, the number of their close relatives, Photorhabdus species, has recently doubled, from four to twenty in the last few years [Interestingly, the genus mierczak  reportedtis spp. . Admitteew years .X. nematophila subspecies were raised to the species level via further description using a polyphasic technique, i.e., X. poinarii, X. beddingi, and X. bovienii [Xenorhabdus species as EPN symbionts continued relying on sequence data of single gene sequences until the year 2007, when a compilation of 20 Xenorhabdus species was published [Xenorhabdus species were identified via multi-locus sequence analysis and whole genome sequence data. Some recent references, e.g., [Xenorhabdus includes the following 26 species: X. beddingii, X. budapestensis, X. ehlersii, X. cabanillasii, X. doucetiae, X. eapokensis, X. griffiniae, X. bovienii, X. hominickii, X. innexi, X. indica, X. japonica, X. ishibashii, X. khoisanae, X. koppenhoeferi, X. magdalenensis, X. kozodoii, X. mauleonii, X. nematophila, X. miraniensis, X. poinarii, X. szentirmaii, X. romanii, X. stockiae, X. vietnamensis, and X. thuongxuanensis. Nonetheless, other novel species are still in the pipeline. For example, X. lircayensis linked to its partner S. unicornum was recently described [T demonstrated consistent differences from the other described Xenorhabdus species. In addition, the authors presented phylogenetic approaches to prove that VLST is a novel strain of X. lircayensis. While they presented phylogenetic relationship placing X. lircayensis within the genus Xenorhabdus based on the 16S rRNA gene analysis, a phylogenetic reconstruction was also established to include X. lircayensis based on core genome sequences [Xenorhabdus are anticipated to be raised. This expectation is based on both their importance, especially against many pests/pathogens, and the recent physiological, biochemical, and molecular techniques to identify them as mutualistic or symbiotic bacteria of Steinernema spp. Therefore, it could be concluded that the progressive increment in the number of novel Xenorhabdus species and strains and new technological methods to characterize them have led to a well-established Xenorhabdus taxa with finer and easier profile to study than before. Consequently, sound genome-constructed phylogenetic trees, enriched with precise sequence comparative studies, could perfectly define their diversity and relationship. These efforts for their characterization and identification should also take part in further research of the related Xenorhabdus spp.-bioactive complexes that can be utilized in agricultural and industrial products [Initially, three bovienii ,20. Idenublished . Thereafs, e.g., ,22, statescribed  to elevaequences . Ultimatproducts ,24.Xenorhabdus have a mutualistic relationship with the entomopathogenic nematodes (EPNs) of the genus Steinernema. The bacteria live symbiotically in the specialized intestinal vesicles of Steinernema. The two partners naturally form an antagonist sharing mainly against their insect hosts. The EPN-third-stage infective juveniles (IJs) conserve the bacteria in their body from the outer environmental stresses until these IJs release them within the insect body. In addition, after EPN infection and depleting the host resources, the IJs vector the bacteria from one susceptible host to another. In turn, Xenorhabdus spp. generate antimicrobial compounds and secondary metabolites into the insect. These materials can not only kill the insect host and prepare the contents of its body to feed the nematodes for their development and reproduction, but also protect the insect cadaver from soil scavengers and saprobes [Basically, the bacterial species in the genus saprobes . During Xenorhabdus is sometimes linked to its counterpart Photorhabdus bacteria, since much similarity is generally found between them in the frame of living. Xenorhabdus and Photorhabdus comprise symbiotic bacteria of EPN-IJs, of the genera Steinernema and Heterorhabditis, respectively. Nonetheless, each of them possesses particular bacterial traits that differentiate its identity. Xenorhabdus spp. survive within a receptacle exhibiting biological specialization at the anterior part of the intestine. Photorhabdus spp. are found at the mucosa of their nematode guts [Steinernema spp.-infective juveniles mature to amphimictic males and females. Therefore, the dyad  must infect their susceptible host to mate then reproduce within the insect hemocele. Heterorhabditis–IJs develop into hermaphrodites  and then, in subsequent generations, to males/females. As a result, a single hermaphroditic–IJ has the merit to infect and reproduce in the host insect. Moreover, the demeanor of bacterial colonization of Xenorhabdus and Photorhabdus differs within their nematode partners [Photorhabdus (bacterium) to tinge the insect host’s body in a reddish tint. Photorhabdus can glow so frequently that its entire infected host usually glows in unlighted sites. To counteract the insect host-immune system, Photorhabdus can modify the lipopolysaccharide to tolerate the influence of the host-derived antimicrobial generation of peptides, but Xenorhabdus disorganizes the creation of these peptides [Photorhabdus species can go through major transcriptional reformation in their host, EPN, intestine. They can stimulate general starvation mechanisms, switch to the pathway of pentose phosphate to comply with nutrition shortage and oxidative stress, cellular acidification to slacken growth, and shape biofilms to firmly last in the nematode intestine for subsequent transmission to the hemolymph of the insect [Mentioning ode guts ,26. Steipeptides . Both gepeptides . Photorhe insect . These be insect ,18.Xenorhabdus bacteria have essential services in the biocontrol processes against pests [S. carpocapsae–X. nematophila and S. puntauvense–X. bovienii indicated important metabolic shifts related to the rearing conditions as well as bacterial (presence versus absence). Nematode genes involved in the production of amino acid, carbohydrate, and lipid were downregulated as IJs were cultured in vitro, in the presence or absence of the symbiotic bacteria [S. carpocapsae and S. puntauvense displayed a unique mechanism to adjust their virulence when their symbionts were absent. Both axenic nematode species showed a differential manifestation of the toxic protein that they secrete, i.e., absence of their symbiotic bacteria raises the expression of their secreted venom protein. The study suggested that IJs of these nematode species may have a unique mechanism to adjust infecting their hosts in the absence of their Xenorhabdus partners [It is well established that st pests . They, vbacteria . The dowpartners . This doSteinernema–bacterial symbiont specificity and their coevolution have been thoroughly studied for many involved axenic (free of bacteria) and monoxenic (having a Xenorhabdus species) Steinernema species [Steinernema species can presumably set up symbiosis with only one species of Xenorhabdus, any of numerous Xenorhabdus species are able to associate with several Steinernema. On the contrary, the symbiotic Heterorhabditis–Photorhabdus associations are more adaptable as many species of each partner can engage in symbiotic relationships with multiple species of the other partner [Xenorhabdus bacteria between different Steinernema species, relying on the species of Xenorhabdus–Steinernema pair used. However, such switching in the bacterium–nematode pair may have its pros and cons. On the positive side, associations of Steinernema species with new Xenorhabdus partner may validate colonization of novel niches or expand one by offering considerable fitness benefits [Steinernema species. On the negative side, the Xenorhabdus partner switching frequently has a harmful effect on the Steinernema host in terms of a reduction in their fitness, reproduction, and symbiont carriage as well as virulence. For example, Xenorhabdus bovienii is the native symbiotic bacteria of S. feltiae. However, using X. nematophila strain HGB315, not the native symbiont of S. feltiae, this nematode species developed and turned into gravid much faster at approximately 4 days on X. bovienii (versus 5–6 days on X. nematophila HGB315) post-seeding [Steinernema host diversity substantially impacts coadaptation between various Xenorhabdus–Steinernema partners [Xenorhabdus strains based on phenotypic analysis. A group included bacterial strains that can commonly grow at 35–42 °C, while the other group included Xenorhabdus strains that grow below 35 °C. Hence, Xenorhabdus bacteria may be adapted to temperate, subtropical, or tropical regions. They are also differently impacted by the growth temperature of their Steinernema host [Steinernema–Xenorhabdus complex, we must mention that recent references, e.g., [Steinernema–Xenorhabdus complex as partners for the mutualism is still effective. Thus, it can bode well for more investigations concerning their distribution in diversity and space [ species . While a partner . These f partner . However partner . These a partner  concludebenefits ,33. Thes-seeding . These d-seeding . Eventuapartners ,36 for tpartners  could idema host . Moreoveema host  as well ema host . With ths, e.g., , still ind space . Moreovend space  to detecnd space . Howevernd space ,42,43,44Photorhabdus species, all the species of Xenorhabdus are exclusively linked symbionts to the Steinernema spp.–IJ stage [Photorhabdus, materialized in P. asymbiotica as a human pathogen in addition to infecting insects [Xenorhabdus. None of the Xenorhabdus bacteria were found in free-living order in nature; therefore, they had formerly boosted doubts concerning their ability to survive and infect pathogens/pests without the EPN partner.Similar to their near relatives, IJ stage . The exc insects , is not Steinernema–Xenorhabdus partnerships, to attack and kill numerous arthropod pests, have been marketed and utilized as biocontrol agents [Steinernema–Xenorhabdus complex, Xenorhabdus host range is surely limited to the ability of the IJs to locate and infect the host. This is a prerequisite for the development and multiplication of Xenorhabdus spp. to achieve high levels of cells within the host. Factually, both mutualists, Steinernema and Xenorhabdus, can generate bioactive compounds to kill the invaded host [Xenorhabdus rate of growth is tightly related to the time needed to kill the insect host. Clearly, Xenorhabdus spp. are quite virulent pathogens of a broad range of pests/pathogens including insects, fungi, bacteria, protozoa, and nematodes [Traditionally, various l agents , with inl agents ,47,48,49ded host ,41. Therematodes .Xenorhabdus bacteria to live in fresh water and in soil for 6 days has surely opened a new avenue with fixed timeframe for their further biocontrol usages, apart from their mutualistic Steinernema [Xenorhabdus chromosome, having numerous genes that encode various antibiotics, insecticidal protein toxins, enzymes, and bacteriocins, were investigated [Xenorhabdus strain may generate a variety of antifungal and antibacterial compounds. Some of its compounds are active against insects, protozoa, nematodes, and cancer cells, too [X. nematophila strains showed insecticidal activity against representative pests of three insect orders; the cabbage white caterpillar Pieris brassicae (Lepidoptera: Pieridae), the mosquito larva Aedes aegypti (Diptera: Culicidae), and the mustard beetle Phaedon cochleariae (Coleoptera: Chrysomelidae) [Xenorhabdus species/strains to kill/inhibit the growth of the intended pest or pathogen. These variations are based either on the ability of each Xenorhabdus species/strain to generate effective metabolites or the relative susceptibility/tolerance of the targeted pest/pathogen. These differences are found not only between Xenorhabdus species/strains, but surely exist to varying degrees between bacterial species/strains belonging to different genera. Photorhabdus luminescens showed more suppressing effect against Plutella xylostella (the diamondback moth) than that shown by [Xenorhabdus nematophila and P. luminescens induced 40% and 60% mortality of P. xylostella pupae, with LC50 values of 5.5 × 105 and 5 × 104 cells/mL, respectively. This difference does not mean the constant superiority of Photorhabdus over Xenorhabdus bacteria. On the contrary, the cells of X. budapestensis strain DSM 16342, X. szentirmaii strain DSM 16338, and P. luminescens ssp. laumondi strain TT01 could induce 100%, 88%, and 79.3% mortality rates of the locust bean moth Ectomyelois ceratoniae  larvae, respectively, at three days’ post-exposure [E. ceratoniae to protect pomegranate (Punica granatum) fruit cultivars [Meloidogyne javanica than X. bovienii where the nematode-induced mortalities were 88, 94.7, and 67.7%, respectively, at 24 h post-application. To explain the difference in nematode-mortality percentages, the authors [X. bovienii are different from those of X. nematophila and P. luminescens. Furthermore, among nine bacterial symbionts of EPNs, X. nematophila HB310 showed the best insecticidal activities against Locusta migratoria manilensis (Orthoptera: Acrididae)-fourth instar nymph [50 of the strain HB310 culture broth against adult, fourth instar nymph, and second instar nymph were 6.24 × 105, 4.90 × 105, and 2.97 × 105 CFU/mL at 96 h, respectively. In addition, the LT50 of its culture broth were 64.34, 52.59, and 51.48 h, respectively. Moreover, this strain exerted antifeeding activity to locust where the median antifeedant concentrations (AFC50) of its culture broth were 3.16 × 105, 2.36 × 105, and 1.15 × 105 CFU/mL at 48 h, respectively. The authors [Xenorhabdus species could be more effective against pests/pathogens than others related to the same genus. The adults of Drosophila melanogaster showed resistance to X. innexi [Manduca sexta larvae, while X. nematophila proved effective against the two insect species [Discovering the competency of inernema . Consequinernema ,50,51,52stigated ,53, and stigated ,42,49,54lls, too . All tesmelidae) ,44,55,56exposure . In addiultivars  were mor authors  reportedXenorhabdus species/strain and/or to increase its potency is to introduce other antagonists in combination with Xenorhabdus bacteria and/or their bioactive compounds. This approach can establish and boost the efficacy of the introduced organism, too. Clearly, synergistic activity to kill the beet army worm Spodoptera exigua could be obtained by mixing growth media supernatants of Xenorhabdus bacteria with B. cereus or B. thuringiensis spores. In this case, while the supernatant of Xenorhabdus bacteria could exert its impact on the insect hemocoel, the Bacillus cells were able to perforate the insect midgut epithelium [B. thuringiensis (Bt) spores and culture broth of X. nematophila (Xn). Although this product demonstrated high toxicity, it has also some modification to widen its efficacy against a diverse insect pest spectrum. Their tests centered on increasing “Bt-Plus” toxicity against a semi-susceptible insect, S. exigua, via adding Xn metabolites. Given the fact that Xn metabolites, benzylideneacetone (BZA) and oxindole (OI), can boost the Bt insecticidal activities, adding each of them (OI or BZA) could significantly enhance Bt-Plus pathogenicity. Moreover, when the freeze-dried Xn culture broth was included into Bt-Plus, a much smaller amount could suffice to raise the toxicity relative to the amount of BZA or OI. High-performance liquid chromatography analysis revealed that there were more than 12 unidentified X. nematophila metabolites in Xn culture broth. Therefore, they [X. nematophila metabolites, not solely OI and BZA. Likewise, a Xenorhabdus species could induce high mortality of S. exigua third-instar larvae but its pathogenicity was much less for the fifth-instar larvae. Seongchae and Yonggyun [B. cereus, a gut symbiont needed to optimize S. exigua development. To enhance the Xenorhabdus species pathogenicity in the fifth instar, the bacteria should be delivered into the hemocoel. Thus, the authors utilized B. thuringiensis aizawai (Bt) as a synergist to back entry of the bacteria from the insect gut lumen into its hemocoel by disrupting the S. exigua gut epithelium. As a result, the applied bacterial mixture was highly synergistic against the S. exigua fifth-instar larvae. This synergism was proved via the successful infection of X. sp. or Bt in the insect hemocoel. Therefore, Xenorhabdus bacteria can be used to kill S. exigua by oral treatment in a mixture with Bt [An obvious technique to circumvent the lack of appropriate efficacy of ithelium ,62. LateYonggyun  speculat with Bt .Xenorhabdus bacteria and/or heterologously expressed in other bacterial systems [Xenorhabdus can be genetically engineered to enhance the potential virulence of its nematode host against more resistant herbivore pests [X. bovienii-derived protease inhibitor protein in tobacco (Nicotiana tobacum cv. Samsun NN) plants could increase tolerance against green peach aphid Myzus persicae (Hemiptera: Aphididae). Fusion proteins of a protease inhibitor from X. bovienii strain PIN1 and green fluorescent protein (GFP) were expressed in the plant [M. persicae was examined by feeding neonate aphids on three independent homozygous lines. When nymphs were fed on PIN1-GFP-expressing plants, no impacts on insect survival were noticed, but fecundity and average insect weight and were considerably decreased. The aphid biomass was reduced by 30–35% relative to those reared on control plants. The effects of PIN1 on M. persicae were tightly related to the decrease of total protease activities of whole insect extracts and the leucine aminopeptidase. Additionally, an elevation in polyphenoloxidase activity was apparent in PIN1-GFP-expressing plants. These results demonstrated that the transgenic expression of PIN1 in tobacco increased plant tolerance against aphids. The study disclosed Xenorhabdus bacteria as other valuable resources of protease inhibitors which can be engineered into plants for insect pest management [In other investigations, other insecticidal toxic proteins were purified from  systems ,64,65. Ire pests . Differehe plant . The effnagement .Xenorhabdus bacteria—such as the ability to disturb the induction of insect-derived antimicrobial peptides, adjusting and fitting to the settings, particularly under stresses, and global spread with a treasure trove of beneficial materials and compounds  was noted at X. nematophila concentration of 4 × 106 cells/mL. Only within 2 h post-application could maximum X. nematophila cells in broth (95%) penetrate into the G. mellonella larval body. Moreover, when the bacterial toxic secretion was dried and then dissolved either in water or broth, it also proved to be effective. On the other hand, the efficacy of bacterial toxic secretion was declined due to storage. Because the bacterial cells can enjoy a free-living lifetime and invade the insect hemocoele without their nematode partner, the authors confirmed that the Xenorhabdus cells, or their impactful secretions, can be utilized for pest control. Therefore, they [X. nematophila or its toxic secretion can be utilized as a significant component of integrated pest management (IPM) programs against Galleria.Obviously, the aforementioned traits of the s e.g., ,41,42)—a,42—aXenos  culture can assure Koch’s postulates [Xenorhabdus bacteria may have wide host range of insect pests. For example, 122 strains of symbiotic bacteria associated with 23 EPNs were gathered from various Chinese localities [Ostrinia furnacalis larvae. One of the strains, however, Xenorhabdus sp. SY5, with determined partial toxin gene sequence, exhibited strong insecticidal activity to a variety of economically significant agricultural pests. Their species comprised Plutella xylostella, Ostrinia furnacalis, Tenebrio molitor, S. exigua, and Mythimna separata. The strain isolated from Steinernema sp. SY5 appeared to have seven purified toxins based on DEAE-52 column chromatography. These toxins exhibited, to a certain extent, growth inhibition and/or insecticidal activity to these insect species. The authors [Usually, re-extraction of the stulates ,66. Althstulates ,52,66, pcalities . These e authors  stressedXenorhabdus spp. still possesses much that has not been discovered yet, for controlling wide categories of many pathogens. In this respect, Hajihassani et al. [X. bovienii and X. szentirmaii metabolites for the root-knot nematode (RKN) Meloidogyne incognita control on cabbage roots in two environmental conditions. At-plant applications of Paecilomyces lilacinus strain 251 (MeloCon WG) and secondary metabolites of Burkholderia rinojensis strain A396 (Majestene) and oxamyl (Vydate) were used for comparison. In the greenhouse, X. szentirmaii and Vydate at 5 DBP had a lower (p < 0.05) root gall rating than the untreated control. Vydate and all metabolite treatments showed significantly lower root galling relative to Majestene, MeloCon, and the control. In addition, the metabolites and Vydate decreased (p < 0.05) RKN egg counts per gram of root compared to the other treatments in the greenhouse. No differences were observed in the egg count between Vydate and the metabolites. At-plant and 5 DBP applications of X. bovienii and X. szentirmaii at decreased the total egg count relative to Majestene and the control in the greenhouse. Thus, the natural metabolites generated by the two Xenorhabdus species can control M. incognita regardless of application timing and are suggested as a potential alternative to nematicides in organic production systems [X. lircayensis, identified using the whole genome, was evaluated on a population of the plant-parasitic nematode Xiphinema index [X. lircayensis were resuspended in phosphate-buffered saline (PBS) and set to 1 × 106 and 1 × 107 CFU mL−1 for laboratory and semi-field assays, respectively. Cell bacteria (1 × 107 CFU mL−1) were applied in the semi-field assay by 30 min dipping grapevine roots in the bacterial suspension. Afterward, these plants were established in 5 L pots filled with naturally X. index-infested soil and immediately inoculated with 350 mL of the same X. lircayensis suspension. The nematicidal effects of X. lircayensis suspension appeared at 24 h post-inoculation but attained full (100%) X. index mortality after 72 h exposition (p < 0.001) in laboratory assays. In addition, under semi-field conditions, X. lircayensis suspension significantly (p ≤ 0.05) reduced X. index populations. While the study recommended X. lircayensis as a good candidate for further assesses in field conditions, additional analyses must be performed to set the metabolites, enzymes, and mode of action for its nematicidal aptitude [X. nematophila and P. laumondii on another plant pathogenic category represented by the fungus Botrytis cinerea growth and compared the activity of bacteria isolated from a bio-fermenter with the commercial B. amyloliquefaciens . The UF and CFS of X. nematophila suppressed about 95% and 80% of B. cinerea growth, respectively, while both UF and CFS of P. laumondii inhibited only about 40%. These data showed the potential of CFS and UF of X. nematophila for B. cinerea control.It is likely that the arsenal of i et al.  assessed systems . In addima index . Supernaaptitude . Vicenteaptitude  tested tX. bovienii metabolite treatment was comparable to fenbuconazole  in decreasing Fusicladium effusum sporulation on pecan (Carya illinoinensis) terminals. X. bovienii metabolite and broth treatments suppressed development of lesions brought about by Phytophthora cactorum (using pecan tree leaves maintained on agar). The bacterial metabolite treatment was also toxic to Armillaria tabescens, another important pathogen but of peach (Prunus persica) trees, especially in the southeastern United States [X. nematophila generates many metabolites during growth and multiplication. Only one of these secondary metabolites (xenocoumacin 1) proved to have a robust antifungal activity for controlling Rhizoctonia solani [Botrytis cinerea [Alternaria alternate [Phytophthora species, etc. [Xenorhabdus species, which are available or are likely to befit broadly soon, are able to control other pests and diseases. Recently, Xenorhabdus budapestensis strain C72 showed remarkable suppressing effect on spore germination and mycelial growth of the fungus Bipolaris maydis which causes the Southern corn leaf blight [X. szentrimaii had the highest fungicidal effect on mycelium growth of Sclerotinia sclerotiorum. They reported that X. szentrimaii produces volatile organic compounds that inhibit S. sclerotiorum growth and/or its consequent generation of sclerotia.In another study , X. bovid States . These ra solani , Botryti cinerea , Alternalternate , severales, etc. ,74,75,76f blight . The relf blight . Chacón-f blight  reportedXenorhabdus are still in progress [Xenorhabdus bacterial dosage [The discovery and cloning of additional useful compounds from progress ,43,54,57l dosage ,42.Xenorhabdus bacteria are typically famous for killing their hosts via toxemia/septicemia, within the form of the normal Xenorhabdus–Steinernema complex [Steinernema species carrying specific Xenorhabdus strains can invade a single insect, Xenorhabdus spp. are also engaged in competition with both related strains and nonrelated gut microbes of the insect host [Xenorhabdus having the capability to kill the insect host, can explain why Xenorhabdus spp. produce a treasure trove of diverse insecticidal and antimicrobial compounds. Moreover, Ciezki [X. bovienii and X. nematophila can generate R-type bacteriocins (xenorhabdicins) that are specifically active towards different Xenorhabdus and Photorhabdus species. The latter author stressed that xenorhabdicin activity could be predictive of competitive results between two Xenorhabdus strains, while other determinants, besides xenorhabdicins, were mainly included in the competitive success between the other Xenorhabdus strains. Thus, Ciezki [Xenorhabdus antibiotics could define the output of interspecies competition in a natural host environment.The  complex . Howeverect host . This co, Ciezki  found th, Ciezki  demonstrXenorhabdus-derived compounds in industrial products stems from not only their abundance, but also their qualities that enhance their functions. Initially, standalone pathogenicity trials of Xenorhabdus bacteria and/or their released materials usually start with their direct injection into the haemocoel of insects via artificial means [Xenorhabdus protein toxins ordinarily have oral or/and injectable toxicity to insects. Xenorhabdus-derived compounds have a variety of modes of action that have been reviewed [Xenorhabdus–dithiopyrrolone derivatives (comprising the two metabolites xenorhabdins and xenorxides) is inhibition of RNA synthesis [Xenorhabdus–indole-containing compounds could show additional mechanism via weak phospholipase A2 inhibitory effects. This latter, phospholipase A2, is necessary for producing eicosanoids. Eicosanoids have substantial role for activating the insect-immune response via mediating and modulating hemocyte behavior [X. nematophila can suppress the immune response of the insect host to be more vulnerable to microbial infection. Xenorhabdusbudapestensis has two antimicrobial peptides, GP-19 and EP-20, with wide-spectrum antimicrobial activity against bacteria and fungi [The mounting ambition to harness al means ,66. Xenoreviewed ,69,79. Tynthesis . Howeverbehavior . Thus, Dbehavior  assumed nd fungi . The firnd fungi .Xenorhabdus species/strains has been uncovering the ability of these bacteria to produce numerous secondary metabolites. Thus, it can contribute to comprehensive examination of the molecular basis underlying the biological control activity of this Xenorhabdus strain [Xenorhabdus bacteria. The main antimicrobial materials comprise ribosomal-encoded benzylideneace-tone [Xenorhabdus lipopeptides [Xenorhabdus doucetiae and X. mauleonii and known as xenoamicin, are tridecadepsipeptides with hydrophobic amino acids [X. doucetiae DSM 17909 revealed that xenoamicins are encoded by five non-ribosomal peptide synthetases (NRPSs), XabABCD, and an aspartic acid decarboxylase (XabE). Due to its hydrophobic characteristics, xenoamicin can interact with the host–cytoplasmic membrane. Nevertheless, no antifungal or antibacterial activity has been listed for xenoamicin A, which displays a different mechanism. Xenoamicin A has weak cytotoxic and anti-protozoal activities [X. indica, has supplemental fatty acid chain linked to one of the amino acids [X. nematophila [X. szentirmaii and Xenorhabdus sp., strain PB30.3 [Plasmodium falciparum (protozoan parasite of humans) and have some activity against Trypanosoma brucei rhodesiense and Trypanosoma cruzi (parasites of many vertebrates). Szentiamide possesses a weak cytotoxic activity against Galleria mellonella hemocytes. Xenobactin has no cytotoxic activity; yet, it is active against Micrococcus luteus. This antibacterial activity is mostly due to its hydrophobic status where it probably targets the bacterial cell membrane [Xenorhabdus and Photorhabdus [Xenorhabdus species but Photorhabdus species do not produce fabclavines, except for P. asymbiotica [Photorhabdus species  did not show antiprotozoal activity [Xenorhabdus species are xenorhabdins, xenocoumacins, and PAX peptides. Thus, the tested Xenorhabdus species were more effective against the serious human protozoal parasites Entamoeba histolytica, Acanthamoeba castellanii, Trichomonas vaginalis, Trypanosoma cruzi, and Leishmania tropica [Xenorhabdus-derived toxins will uncover certain variations among bacterial strains regarding toxicity to these pests. Various features and details concerning the mode of action, structure, and putative function of the Xenorhabdus-bioactive compounds in the process of infection have been clarified [Complete genome sequencing of various s strain . Variousace-tone  xenocin ace-tone ,86, and ace-tone , fabclavace-tone , xenocouace-tone , nematopace-tone , rhabdopace-tone , and peppeptides . Knowingpeptides ,41. For no acids . The gentivities . The secno acids . The pepno acids . The thiatophila , is antin PB30.3 ,97. Bothmembrane . Eventuaorhabdus ,78. Fabcmbiotica . This caactivity . On the activity . Other a tropica . Furtherlarified ,79.Xenorhabdus bacteria with their beneficial functions are known  possessing genes that are responsible for encoding low-molecular-weight secondary metabolites/toxins with various activities against many pests/pathogens, e.g., insects [Xenorhabdus bacteria in pest control under laboratory conditions [Xenorhabdus bacteria releases toxins with substantial activities in the insect intestinal epithelium [Xenorhabdus bacteria can have synergistic activity, when added to other biocontrol agents, against serious and intractable insect pests such as the beet army worm [X. ehlersii protein (XeGroEL) is reliable for G. mellonella control [Xenorhabdus bacteria could have acaricidal and antibacterial activities, e.g., X. stockiae PB09 [Xenorhabdus-derived bioactive compounds can offer novel templates for commercial use, obtaining reliable and environmentally safe alternatives to currently unsafe pesticides, e.g., to control phytonematodes [Xenorhabdus nematophila-derived bioactive compounds blocked the feeding of crickets, ants, and wasps [Xenorhabdus-derived metabolites such as oxindole and benzylideneacetone [Xenorhabdus-derived bioactive compounds could be used not only in agricultural sustainable systems but also in pharmaceutical and industrial products. A mixture of Bacillus thuringiensis israelensis (Bti) spores with X. nematophila culture broth containing metabolites has superior efficacy in controlling Aedes albopictus mosquitoes and Culex pipiens pallens. Based on enhanced Bti toxicity in the mixture against the two insect species, a commercial product was created, named “Dip-Kill” [P. temperate temperata and X. hominickii could also boost the toxicity of Bti against culicides [The growing interest in investigating  insects ,42, fung insects ,79, prot insects ,51, bact insects ,80, plan insects ,121, and insects ,122;  and undnditions ); (iii) ithelium ; (iv) Xermy worm ,63; (v)  control ,127;  nd wasps ,132;  and lowest (2.11 ± 0.09 kg tomato fruit/plant) were harvested from X. bovienii  treatment and control, respectively (p < 0.0001). While such results furnish growers with hands-on knowledge for decision-making to determine the financial feasibility of these different RKN control strategies [Xenorhabdus bacteria can suppress infection by plant pathogenic fungi [Xenorhabdus bacterial efficacy on various pests and pathogens associated with tomato, or other PPNs-susceptible plant species, in specific agricultural systems should lead to conclusive and practical results.Several published books and tomes, e.g., ,135,136, species ,99,121. urfgrass . Notablyic fungi , in addiX. nematophila gained not only resistance to the cotton bollworm Helicoverpa armigera (Lepidoptera: Noctuidae) but also tolerance against salt and thermal stresses [1 of genetically engineered crops is challenging and requires further investigations. Such crops may be introduced to growers in rural communities with varying social communities and heterogeneous agricultural systems. On the other hand, there is a great potential of using Xenorhabdus bacteria in pharmaceutical and industrial products. For example, their products can be used to control vector insects of human diseases such as dengue and malaria. In this respect, occurrence of mosquito’s resistance to Bti was documented [Xenorhabdus nematophila-cultured broth could deter the resistance and strengthen toxicity against such vector insects as Aedes albopictus and Culex pipiens pallens (Diptera: Culicidae) [Xenorhabdus bacteria against mosquito-borne arboviruses and other microorganisms of the Plasmodium group.Tomato plants expressing certain genes of stresses . With sucumented . Nonethelicidae) ,133. FurP. luminescens, cell phenotypic variation in X. nematophila strains could be controlled via regular selection of primary variants. Consequently, no trait change was detected in the X. nematophila primary variant after prolonged subculture. The genetic basis for this stability is a merit for the bacterial economical and biocontrol applications of X. nematophila production [Xenorhabdus bacteria have superior production  against the need to preserve traits significant for biocontrol. Furthermore, the fermentation process was recently optimized for xenocoumacin 1 (Xcn 1) production by X. nematophila [X. nematophila fermentation production of Xcn1 was raised to 3.4-fold (234.9 mg/L) and 3.6-fold (249.7 mg/L) at 1.0 and 0.5% L-arabinose concentration, respectively [Xenorhabdus-bioactive compounds.Contrary to oduction  because atophila ,140. As ectively . These iXenorhabdus formulations have been implemented . These formulations have potential to be further developed as commercial products for controlling mushroom mites [X. szentirmaii in a powder form against the fungus Botrytiscinerea. This effect of the antifungal metabolites of X. szentirmaii in a powder form was weaker than its corresponding liquid X. szentirmaii supernatant [Xenorhabdus powder has longer shelf-life and can be easily and economically formulated and applied compared to liquid supernatants, the author suggested that further studies should optimize the processes for the powder formulation to enhance its efficacy. In such a case, the system should possess all factors for success including high levels of Xenorhabdus virulence, favorable pulverization process, proper contact with the tested fungal pathogen, and compatibility with current practices, e.g., temperature.Various lemented  with speom mites . An oppoernatant . BecausePhotorhabdus bacteria, which has the human-pathogenic P. asymbiotica [Xenorhabdus bacteria have never been detected from clinical specimens [Xenorhabdus bacterial studies on their bioactive compounds have somewhat lagged behind those of Photorhabdus bacteria. For instance, the high-molecular-weight insecticidal “toxin complexes” were first identified in P. luminescens strain W14 and subsequently in X. nematophila [Contrary to mbiotica , Xenorhapecimens . Howeveratophila . To concatophila . Yet, thatophila .Xenorhabdus bacteria as biologicals in various sustainable agricultural systems against a wide range of plant pests and pathogens is apparently an attractive and promising approach. This bacterium can be exploited directly (with its nematode partner) or indirectly (without its partner) to neutralize the plant–phytonematode interaction to the advantage of the plant. Such an emerging approach should be tried in earnest in order to control not only PPNs, but also many other plant pests and pathogens within IPM plans. The detection of new Xenorhabdus species and strains globally will occur to widen the pool of these bacterial-related materials that are reliable for managing economically significant pathogens/pests. Developing low-priced techniques for their trade production, formulation, and application may expedite their use in current or future management programs. In such programs, Xenorhabdus bacteria can be used with their Steinernema host or as standalone pesticides for common management strategies. Obviously, these bacteria produce a treasure trove of diverse compounds that can kill many harmful insects and microorganisms, e.g., protozoa, PPNs, fungi, oomycetes, and bacteria. Xenorhabdus bacteria could be applied in various formats for topical application, e.g., pellets, powder, spray, suspension, or supernatant. Furthermore, their toxin genes could be incorporated into transgenic plants to control insect pests. The transgenic plants could also gain tolerance against salt and thermal stress in some cases. Therefore, Xenorhabdus bacteria and their bioactive compounds are highlighted herein; in order to provide a substantial way forward in pest management and crop protection, they should be leveraged to fit into holistic crop management strategies. These may comprise their combination with other additive or synergistic inputs, to enhance their efficacy while blocking, or decreasing, the likelihood of developing pesticide-resistant harmful strains.Accelerated public concerns over the misuse and impact of chemical pesticides on human health and environment create the need to discover alternative methods for management of plant pests and pathogens. Utilizing


Cinnamomum camphora is one of the most commonly used tree species in landscaping. Improving its ornamental traits, particularly bark and leaf colors, is one of the key breeding goals. The basic helix–loop–helix (bHLH) transcription factors (TFs) are crucial in controlling anthocyanin biosynthesis in many plants. However, their role in C. camphora remains largely unknown. In this study, we identified 150 bHLH TFs (CcbHLHs) using natural mutant C. camphora ‘Gantong 1’, which has unusual bark and leaf colors. Phylogenetic analysis revealed that 150 CcbHLHs were divided into 26 subfamilies which shared similar gene structures and conserved motifs. According to the protein homology analysis, we identified four candidate CcbHLHs that were highly conserved compared to the TT8 protein in A. thaliana. These TFs are potentially involved in anthocyanin biosynthesis in C. camphora. RNA-seq analysis revealed specific expression patterns of CcbHLHs in different tissue types. Furthermore, we verified expression patterns of seven CcbHLHs  in various tissue types at different growth stages using qRT-PCR. This study opens a new avenue for subsequent research on anthocyanin biosynthesis regulated by CcbHLH TFs in C. camphora. Cinnamomum camphora is an evergreen tree species with a large canopy and dense shade all year round. C. camphora is one of the most commonly used tree species in landscaping, with high economic, ornamental, and ecological value [C. camphora is a native landscape tree in southern China, and its ornamental traits, such as bark and leaf colors, are important selection targets. Many studies have shown that the color of the bark and leaves of angiosperms depends on the proportion and distribution of four pigments: chlorophylls, flavonoids, carotenoids, and betaine [C. camphora demonstrated that red bark results from an enrichment of anthocyanins [Penicillium digitatum) that causes green mold [al value . C. camp betaine . Our preocyanins . These pocyanins . Compareeen mold .PAL, 4CL, CHS, F3H, F3′H, DFR, LAR, ANR, UGTs, and OMT, have been verified by qRT-PCR, and their expression levels in ‘Gantong 1’ were significantly higher than in their half-sib progeny [The pathways of anthocyanin biosynthesis in plants have been progressively elucidated in recent years. Anthocyanin biosynthesis starts from phenylalanine. After a series of enzymatic catalytic steps, the final products are transported to vacuoles and other parts for storage . The MBWbasic and HLH sub-domains. The basic domain contains 13–18 amino acids at the N-terminal, which binds to the E-box (5′-CANNTG-3) [Arabidopsis thaliana, 183 in Populus trichocarpa, 197 in Pyrus bretschneideri, 115 in Vitis davidii, 91 in Liriodendron chinense, 134 in Camellia sinensis, 118 in Ficus carica, and 95 in Gardenia jasminoides, respectively [Ficus carica, and performed transient expression verification of FcbHLH42, confirming that this gene promotes anthocyanin synthesis and accumulation [Juglans regia genome, 4 of which were involved in anthocyanin synthesis [A. thaliana [DFR) gene in seedlings and pods and participates in the formation of the MBW complex [DhDFR and DhANS in the petals of Dendrobium, thereby modulating anthocyanin production [TfbHLH1 plays a significant role in the regulation of anthocyanin biosynthesis in tulip tepals [PgbHLH may be involved in the determination of differences in flavonoid composition in both flowers and fruits of the pomegranate varieties Wonderful and Valenciana [The bHLH TF family, named after its highly conservative basic/helix–loop–helix domain, is widely represented in plants ,11. The ANNTG-3) . The C-tANNTG-3) . So far,ectively ,21,22. Mynthesis . bHLH TFthaliana . Some bH complex . The traoduction . TfbHLH1p tepals . Arlottalenciana .C. camphora are usually dark green and dark brown, respectively. In contrast, the bark and leaves of the natural mutant C. camphora ‘Gantong 1’ are red to dark red and orange-red to yellow-green, respectively. Hence, the characterization of bark and leaf coloration of ‘Gantong 1’ would significantly contribute to our understanding of the coloring mechanism of C. camphora. The recently released whole genome sequence of C. camphora is a valuable tool to study the bHLH TF family of C. camphora in detail [C. camphora at the genomic level, revealed their potential roles in the regulation of anthocyanin biosynthesis, and provided valuable insights for genetic improvement and molecular breeding focused on the coloration of the ornamental tree C. camphora.The leaves and bark of normal n detail . In thisbHLH TF family members were identified by the Hidden Markov Model (PF00010) in the C. camphora genome. Subsequently, we used the NCBI Batch CD-Search database and SMART to determine the presence of the conserved bHLH domain and remove the redundant sequences without the bHLH domain. Finally, 150 members of the CcbHLH TF family were identified from the whole genome protein file of C. camphora. These genes were named CcbHLH1–150 based on their chromosomal position , followed by chromosome 2 (20) and chromosome 1 (17). Chromosomes 8 and 12 had 7 CcbHLHs each. A total of 143 bHLH TFs were identified in the C. kanehirae genome, which was 7 fewer than the number of CcbHLHs in the genome of C. camphora.In total, 173 position . The 150Then, the physicochemical properties of CcbHLH proteins were computed by using Protparam EXPASY online software. CcbHLH proteins ranged from 86 (CcbHLH073) to 1216 amino acids (CcbHLH091) in length. The molecular weight of CcbHLH proteins ranged from 10.04 kD (CcbHLH110) to 136.68 kD (CcbHLH091). The hydrophilicity of CcbHLH proteins ranged from −1.016 (CcbHLH066) to −0.086 (CcbHLH021), indicating that all CcbHLH proteins are hydrophilic. The theoretical isoelectric points of CcbHLH proteins ranged between 4.49 (CcbHLH032) and 11.98 (CcbHLH086). The instability index of CcbHLH proteins ranged from 37.35 (CcbHLH046) to 86.82 (CcbHLH047), and only CcbHLH008, CcbHLH046, CcbHLH109, and CcbHLH148 proteins had instability indices less than 40, indicating that most CcbHLH proteins may be unstable .A. thaliana (162) were downloaded from the TAIR databases [C. camphora and A. thaliana using MAFFT version 7, a rootless phylogenetic tree was constructed based on 162 AtbHLHs  were classified as “Orphans”. Twenty-eight clades coalesced into 27 subfamilies and 1 “Orphan” subfamily. CcbHLHs were distributed in 26 of these subfamilies, and 2 subfamilies, VI and XV, belonged to the specific family of A. thaliana. The number of members in different subfamilies varied greatly. Among them, Ib was the largest subfamily, with 21 members, followed by subfamilies XII and X, with 16 and 12 members, respectively. The smallest subfamilies, Va, XIII, IVb, and XIV, each had two CcbHLH members. The classification result of the C. camphora bHLH TF family based on the phylogenetic tree illustrates the evolutionary relationship of CcbHLHs.The recently reported numbers of bHLH protein subpopulations in different plant species ranged between 15 and 32 ,30,31. T AtbHLHs . We perfCcbHLH functions  on C. camphora chromosomes 4 and 10, respectively. Among the 150 CcbHLHs, there were 68 pairs of segmentally duplicated genes  cooperate with TT1, PAP1 and TTG1 to regulate biosynthesis of proanthocyanidins and anthocyanidins by modulating the expression of the DFR gene  tissues, including the stem, fruit, root, xylem, leaf, flower, and phloem. A hierarchical clustering analysis found significant differences in CcbHLHs expression patterns (CcbHLHs with relatively high expression levels (FPKM > 10) were observed in the phloem, flower, leaf, xylem, root, fruit, and stem tissues, respectively. Among them, 22 CcbHLHs  showed high expression levels in all tissues (FPKM > 10). CcbHLH042 showed the highest transcript accumulation in the phloem and leaf (FPKM > 150). Further, CcbHLH005, CcbHLH097, CcbHLH120, CcbHLH148, and CcbHLH149 showed the highest expression level in the flower, xylem, root, fruit, and stem (FPKM > 147), respectively. However, 17 CcbHLHs  had very low or no expression in all seven tissues tested (FPKM < 0.5).To reveal the expression pattern of patterns . In partCcbHLH TFs in anthocyanin biosynthesis in C. camphora, we characterized the expression levels of seven CcbHLHs in various tissue types at different stages by qRT-PCR. Three CcbHLHs were identified previously  and four were revealed in this study   peaked in April and May, while expression of CcbHLH022 and CcbHLH134 peaked in May and August. The expression of CcbHLH101 reached its peak in July and August. However, the expression levels of CcbHLH118 showed one peaks in April  . We compin April A. Furthein April B. IntereC. camphora, one of the essential landscaping species of the world, is widely used in street-side greening and to create shade. The colorful leaves, bark, and other ornamental traits are important breeding goals for C. camphora. bHLH TFs are the essential TFs regulating anthocyanin biosynthesis. The function of some bHLH TF family members in anthocyanin biosynthesis has been previously demonstrated in species such as Vitis davidii, Ficus carica L., and Juglans regia L. [bHLH TFs are involved in anthocyanin synthesis in C. camphora (‘Gantong 1’) [bHLHs in regulating the colors of C. camphora tissues remained unclear. The elucidation of genomic characteristics of C. camphora bHLH TFs could help understand the development of this ornamental trait.regia L. ,21,23. WCcbHLH TF family members were identified in C. camphora, which was a similar number of bHLH TFs compared to that in Panax ginseng (169) and tomato (159) [C. camphora was larger than that in Liriodendron chinense (91) [Prunus avium L. (66) [Juglans regia L. (102) [Helianthus annuus L. (183) [Pyrus bretschneideri (197) [bHLH transcription factors were identified in C. kanehirae of the same genus as C. camphora, which was basically similar to C. camphora, indicating the same evolutionary relationship within the same genus. C. camphora is rich in essential oils, which contain terpenoids. The leaf essential oil of C. camphora is considered a contributor to the beneficial properties of this plant [A. thaliana directly binds to the promoters of TPS21 and TPS11 and activates their expression, thereby promoting biosynthesis of (E)-β-caryophyllene and other terpenoids [C. camphora may contain more CcbHLH family members than other species to regulate TPS gene expression.In this study, 150 to (159) ,37. The nse (91) , Prunus  L. (66) , and JugL. (102) , but smaL. (183)  and Pyruri (197) . These dis plant . Terpenerpenoids . TherefoAndrographis paniculata [Panax ginseng [Osmanthus fragrans [A. thaliana [Arabidopsis, the minor subfamily VI was not found in C. camphora, which may be attributed to the loss of genes during evolution. We then adopted the Pires’s classification method to analyze CcbHLHs that did not match Heim’s classification in C. camphora. These members then fell into subfamilies XIII, XIV, and Orphan. The numbers of CcbHLH TFs in subfamilies Ib, X, and XII were the largest, broadly in agreement with the pattern of each corresponding subfamily in A. thaliana. Subfamilies Va , XIII , IVb , and XIV  had two members each, suggesting a relatively slow evolution rate. So far, the biological functions of CcbHLHs still remain unclear. However, according to this study and previous studies on other plant species, we were able to narrow down the number of candidate CcbHLH TFs potentially involved in anthocyanin biosynthesis. For instance, CcbHH001, CcbHH022, CcbHH118, and CcbHH134 belonged to the subfamily IIIf, in which A. thaliana (AtbHLH042) was found, which is known to be involved in anthocyanin synthesis [Ficus carica L. [Freesia hybrida [Chrysanthemum morifolium R. [Vitis davidii [The phylogenetic tree results showed that 150 CcbHLH TFs were divided into 26 subfamilies, which is similar to the number of 26 reported for niculata , 24 in P ginseng , 25 in Ofragrans , and 25 thaliana . Howeverarica L. , FabHLH2arica L. , FhGL3L  hybrida , CmbHLH2olium R. , and VdbCcbHLH gene structures and conserved motifs confirmed the phylogenetic relationships within the CcbHLH TF family. Most CcbHLH members in each subfamily shared similar conserved motifs and gene structures, implying semblable biological functions. We found that motifs 1 and 2 occurred most frequently in the CcbHLH TF family, suggesting that these motifs are major components of the CcbHLH domain with highly conserved DNA-binding capacity [CcbHLH TFs ranged from 1 to 17, which was consistent with the numbers obtained in Panax ginseng [Osmanthus fragrans [CcbHLHs had only 1 exon whereas 4 CcbHLHs had more than ten exons. This result suggests the possible ongoing evolution of C. camphora bHLH genes.Further analysis of capacity . In this ginseng  and Osmafragrans , among wCcbHLH gene family drove its expansion. Similar events were also found in Liriodendron chinense [Ficus carica L. [Pyrus bretschneideri [C. camphora and other species, as well as the Ks analysis of homologous genes in the collinearity block, suggested that the C. camphora genome had undergone three genome-wide duplication events during its evolution [In addition, the chromosomal localization analysis suggested that fragment duplication in the chinense , Ficus carica L. , and Pyrhneideri . Furthervolution .CcbHLH TF family genes. Maize R1, B1, Lc, and Sn were the first bHLH TFs shown to regulate anthocyanin synthesis [R transcription factor, TT8, GL3, and EGL3, were then proven to function similarly in Arabidopsis [CcbHLH001, CcbHLH022, CcbHLH118, and CcbHLH134 were orthologous to AtbHLH42/TT8 and, therefore, they likely regulate anthocyanin synthesis in C. camphora.Mutual analysis of CcbHLH proteins can help to predict the potential functions of the ynthesis ,48,49. Tbidopsis . In thisCcbHLHs through transcriptomic analysis using seven different tissue types. For example, CcbHLH005 was highly expressed in the flowers, whereas CcbHLH042 was highly expressed in the leaves and bark. bHLH TFs from the subfamily IIIf were previously reported to regulate anthocyanin and flavonoid biosynthesis [CcbHLH001, was significantly more highly expressed in the stem and phloem compared to its levels in other tissues. However, the expression levels of CcbHLH022, CcbHLH118, and CcbHLH134 did not show any tissue-specificity. Another three CcbHLHs previously identified to be associated with anthocyanin synthesis showed tissue-specific expression patterns as well, including CcbHLH015 in the xylem and phloem, CcbHLH017 in the xylem, and CcbHLH101 in the stem and phloem. We verified the expression patterns of seven CcbHLHs  in coloration mutant ‘Gantong 1’ at various growth stages and in different tissues by using qRT-PCR. The significantly higher expression of CcbHLH TFs in the bark of ‘Gantong 1’ implied their role in the anthocyanin synthesis. Taken together, the transcriptome and qRT-PCR data indicated that these CcbHLHs may play a crucial part in the regulation of anthocyanin biosynthesis. However, the functions of these CcbHLHs  need to be further verified by transgenesis, the yeast one-hybrid method, and other approaches.The specificity of the expression pattern in different tissue types usually indicates the tissue-specific functions of the gene. Thus, we investigated the expression specificities of several ynthesis . One of C. camphora, were selected by the single plant breeding method. The young leaves of this variety are orange-red or orange, and gradually turn to yellow-green or green after maturity. The young bark is light pink with white spots and bright red after half-lignification, with obvious seasonal changes. In this study, we used three C. camphora ‘Gantong 1’ plants and their half-sib progeny, which were planted in the same plot at the Jiangxi Academy of Sciences  in the same growth environment. All plants had a similar growth trend. ‘Gantong 1’ was the experimental group, and the half-sib progeny was the control group  [http://pfam.xfam.org/ (accessed on 1 October 2022)). We used online websites NCBI Batch CD-Search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi (accessed on 1 October 2022)) and SMART (http://smart.embl.de/ (accessed on 1 October 2022)), to perform domain verification of the bHLH TF family protein sequences, and to exclude CcbHLHs without the bHLH conserved domain. Subsequently, we downloaded the genome data and annotation information of Cinnamomum kanehirae from the NCBI database (https://www.ncbi.nlm.nih.gov/data-hub/taxonomy/337451/ (accessed on 1 October 2022)) and made a preliminary identification using the same method. The annotation information of C. camphora was extracted from the GFF file, and the TBtools package was used to visualize it. The physicochemical properties of CcbHLH TFs were analyzed using ProtParam EXPASY online software (https://web.expasy.org/protparam/ (accessed on 3 October 2022)) [Genome sequencing of 0000000) . The Hidr 2022)) .https://mafft.cbrc.jp/alignment/software/algorithms/algorithms.html (accessed on 5 October 2022)) to perform multiple sequence alignment of the AtbHLH and CcbHLH protein sequences based on default parameters. The alignment results were uploaded to MEGA7 software, and a phylogenetic tree was constructed based on the neighbor-joining method and a bootstrap of 1000. Optional parameters were p-distance and pairwise deletion [https://itol.embl.de/itol.cgi (accessed on 5 October 2022)).We used the online tool MAFFT version 7 (deletion . The evohttp://gsds.cbi.pku.edu.cn/ (accessed on 8 October 2022)) and MEME (https://meme-suite.org/meme/ (accessed on 8 October 2022)) to analyze the structures and conserved motifs of CcbHLHs, respectively, on the basis of 150 CcbHLH TFs cDNA sequences with corresponding genomic DNA sequences. The number of conserved motifs was set at 20. The results were visualized with TBtools [We used online software GSDS 2.0 ), and the collinearity analysis was performed using MCScanX software [Ka and Ks values were analyzed with TBtools.The chromosomal localization, length, and density information were extracted from the GFF file of software . The Ka https://cn.string-db.org/ (accessed on 15 October 2022)) to query A. thaliana protein sequences and predict protein interaction networks with 150 CcbHLH protein sequences as references [We used the online website STRING (ferences .C. camphora (‘Gantong 1’) [CcbHLH TFs. The expression levels of CcbHLHs were estimated as kilobases per million (FPKM) reads. Tbtools software was used to visualize transcriptome FPKM data and draw gene expression heat maps based on log2(FPKM).In our previous study, we performed RNA-Seq on seven different tissues  of tong 1’) . These dCcbHLHs candidate TFs for anthocyanin biosynthesis were detected by qRT-PCR using the leaf and bark cDNA of C. camphora (‘Gantong 1’) and the control group, with leaf and bark as templates. Sample RNA was extracted using an RNA extraction kit  and reverse-transcribed into cDNA, which was used as a template for qPCR analysis . The primers were designed using Primer Premier 5.0 software with Tm values ranging from 58 °C to 61 °C and amplified fragments ranging from 100 to 200 bp  potentially involved in anthocyanin biosynthesis in C. camphora. Transcriptional analysis revealed the expression pattern of CcbHLHs in a coloration mutant C. camphora ‘Gantong 1’. This study opens a new avenue for subsequent research on the functions of CcbHLHs in C. camphora.This study is the first comprehensive and systematic analysis of bHLH TFs in the


Amorphophallus konjac). However, the pangenome and genomic plasticity of the konjac soft rot pathogens is little explored. In this study, we reported the complete genome sequences of 11 bacterial isolates that can cause typical soft rot symptoms in konjac by in vitro and in vivo pathogenicity tests. Based on in silico DNA–DNA hybridization, average nucleotide identity and phylogenomic analysis, all 11 isolates were determined to be Pectobacterium aroidearum. In addition, synteny analysis of these genomes revealed considerable chromosomal inversions, one of which is triggered by homologous recombination of ribose operon. Pangenome analysis and COG enrichment analysis showed that the pangenome of P. aroidearum is open and that accessory genes are enriched in replication, recombination, and repair. Variations in type IV secretion system and type VI secretion system were found, while plant cell wall degrading enzymes were conserved. Furthermore, sequence analyses also provided evidence for the presence of a type V secretion system in Pectobacterium. These findings advance our understanding of the pathogenicity determinants, genomic plasticity, and evolution of P. aroidearum.Bacterial soft rot is a devastating disease for a wide range of crops, vegetables, and ornamental plants including konjac ( Pseudomonas, Bacillus, Burkholderia, Pantoea, Enterobacter, Klebsiella, Leuconostoc and Clostridium , a perennial plant belonging to the family Araceae, is widely grown as a cash crop in tropical and subtropical Asian countries such as China, India, and Japan. The konjac glucomannan (KGM), extracted from the corm, is a water-soluble polysaccharide (dietary fiber) with diverse applications in food science and nutrition, biotechnology, pharmacology and fine chemicals  and phylogenomic analysis, new studies have illustrated inconsistencies between established taxonomies and evidence from completely sequenced isolates such as subspecies of P. carotovorum , antimicrobial compounds, and CRISPR-Cas systems, whereas the plant cell wall degrading enzymes (PCWDEs) are highly conserved  medium . BrieflyEleven pathogenic strains (per isolate per sample) were selected and then sequenced. Genomic DNA extraction, sequence library construction and sequencing were conducted at the Beijing Novogene Bioinformatics Technology Co., Ltd. Briefly, total DNA from each isolate at the exponential stage was extracted and assessed using agarose gel electrophoresis and Qubit 2.0 Fluorometer (Thermo Scientific). After library construction, sequencing was performed using a Nanopore PromethION platform and an Illumina NovaSeq PE150 platform. For most bacterial isolates, a hybrid assembly pipeline was conducted by Unicycler v0.4.8 using both Illumina reads and long reads . The genisDDH was estimated via the Genome-to-Genome Distance Calculator 3.0 web server using the recommended formula 2Pairwise average nucleotide identity (ANI) values were calculated by a Python module pyani v0.2.11 using ANIm method.Pectobacterium were retrieved from GenBank in May 2021.P. aroidearum assembly sequences began with the gene dnaA, bacterial synteny was evaluated and visualized by multiple whole-genome alignment using progressiveMauve  models based on core-gene alignment (A maximum-likelihood (ML) tree for the lignment . The Newlignment  and MEGAlignment .Pectobacterium spp. and P. aroidearum, respectively  were detected by uploading the protein sequences to TXSScan/MacSyFinder , 2016. SPectobacterium and Dickeya were downloaded from the UniProt database and used for building a reference database. BLAST+ (v2.6.0) was applied to find putative PCWDEs in 14 P. aroidearum genomes using an E-value threshold . Only hits with at least 35% identity and 50% query coverage were kept. If a protein annotation from BLAST+ results was not consistent with that derived from eggNOG-mapper (v2.1.4), this protein annotation was examined manually.PCWDE proteins of the genera Genomic islands (GIs) were predicted using the IslandViewer 4 webserver.A. konjac tubers with foul-smelling rot symptoms were collected from Qujing, Yunnan Province and Ankang, Shaanxi Province, China. After bacterial isolation and purification, a total of 11 isolates were shown to cause typical soft rot symptoms on konjac tubers in vitro (P. aroidearum (NR_159926), which is consistent with previous findings  Lam.), jícama (Pachyrhizus erosus (L.) Urb.), yacón  H.Rob.) and taro (Colocasia esculenta (L.) Schott), which has not been reported  are incorrectly named.To further confirm the taxonomic status and investigate the genomic diversity of konjac soft rot pathogens, whole-genome sequencing was performed for the 11 strains using a Nanopore PromethION platform and an Illumina NovaSeq platform with more than 100x coverage depth . After hubclades . Notably species . This re species . Sequenc species . Taken tP. aroidearum strains, including genome size, GC (guanine-cytosine) content, number of protein-coding sequences (CDS), and number of RNA genes, were quantified  to 5,057,072 bp (QJ003). GC content varies between 51.6% and 51.9%. The highest number of putative CDS was observed in AK042 , whereas the lowest number of CDS was found in QJ311 . In addition, the number of tRNA, rRNA and CRISPRs  is the same among our 11 genome assemblies. We found no evidence of plasmids in P. aroidearum.Overall genomic features of antified . The lenP. aroidearum isolates  including transposons, insertion sequence (IS) elements, and prophages . The oriPectobacterium, the 64 complete genomes from 9 Pectobacterium spp. including our 11 assemblies were used to identify the core- and pan-genomes. The number of core genes was 2,228, which only accounts for 11.31% of the pangenome, which contained a total of 19,698 genes , species-core genes (1347) and accessory genes (729). Enrichment analysis based on the COG (Clusters of Orthologous Groups of proteins) database showed that genus-core genes are overrepresented in categories associated with essential life activity such as energy production and conversion, the transport and metabolism of amino acid, nucleotide and lipid, and translation, ribosomal structure and biogenesis . The speP. aroidearum genomes to identify and compare these determinants of pathogenicity. All six secretion systems (types I–VI) were detected using the program TXSScan. Type I secretion system (T1SS), type II secretion system (T2SS), type III secretion system (T3SS) and type VI secretion system (T6SS) were conserved in all tested P. aroidearum strains (P. aroidearum genomes (P. aroidearum L6 has two copies of each T4SS subtypes (typeG and typeT) that are classified according to different mating pair formation complexes  play a key role in the interaction between soft rot bacteria and host plants. We mined the 14  strains . However genomes . Notablyxes MPF; . For T6Siderably .Pectobacterium spp. is still inconclusive  of Bordetella pertussis was used as a query to search against protein database of QJ036 using BLASTP. Domain analysis of the three BLASTP hits  shows that each contains a signal sequence, a haemagg_act domain and a filamentous hemagglutinin repeat region  in nclusive , two subnclusive . Domain nclusive . In addihia coli . These rt region . As expeP. aroidearum strains examined in this study. In total, 21 PCWDEs genes were found in each strain including two cellulase genes , one oligogalacturonate lyase gene (ogl), eight pectate lyase genes , two pectin acetylesterase genes , one pectin lyase gene (pnl), two pectinesterase genes , three polygalacturonase genes , two rhamnogalacturonan lyase genes  and one protease gene , which may facilitate microbial adaptation by disproportionately encoding factors involved in virulence or antimicrobial resistance (P. aroidearum isolates were identified using the online webserver IslandViewer 4 (P. aroidearum isolates with similar GIs distribution patterns tended to be from the same sampling site (Genomic islands (GIs) are clusters of consecutive genes likely acquired sistance . Here, GViewer 4 . The locing site . In addiing site . Howevering site . The proing site .P. carotovora subsp. carotovora and P. chrysanthemi, with P. chrysanthemi acting as the major pathogen , which indicates some variation in virulence-related genes among Pectobacterium species. As more genomes of Pectobacterium species become available, it will be important to directly compare the core genome of each species. Interestingly, our comparative genomic analyses strongly suggest that strains including P. carotovorum subsp. carotovorum PC1, P. carotovorum subsp. carotovorum PCCS1 and P. carotovorum subsp. carotovorum PCC21 are misnamed. Instead, P. carotovorum subsp. carotovorum PC1 and P. carotovorum subsp. carotovorum PCCS1 should be classified as P. aroidearum.The development of next-generation sequencing technologies (NGS) has made the pangenome a new tool for analyzing pathogenic bacteria . To date species . The corE. coli, a total of 110 rearrangement events including 19 inversions were detected, and about 70% of rearrangements were associated with recombination between insertion sequence (IS) elements  and the National Natural Science Foundation of China (31860057 and 31901468).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Ci) was assessed using a Windkessel model applied to blood pressure waveforms (finger photoplethysmography) and corresponding middle cerebral artery blood velocity or brachial artery blood velocity waveforms (Doppler ultrasound). Data were analyzed during a 5-min baseline period (10 waveforms) under control conditions and during distinct sympathetic blockade , cholinergic blockade , and myogenic blockade . In experiment 1, phentolamine increased Ci similarly in the cerebral vascular bed (131 ± 135%) and forearm vascular bed . In experiment 2, glycopyrrolate increased cerebrovascular Ci (72 ± 61%) and forearm vascular Ci (74 ± 64%) to a similar extent (P = 0.88). In experiment 3, nicardipine increased Ci but to a greater extent in the cerebral vascular bed (88 ± 88%) than forearm vascular bed . Therefore, adrenergic, cholinergic, and myogenic mechanisms contribute to the regulation of cerebrovascular and forearm vascular compliance. However, myogenic mechanisms appear to exert more specific control over vascular compliance in the brain relative to the forearm.Increasing evidence indicates that cerebrovascular compliance contributes to the dynamic regulation of cerebral blood flow but the mechanisms regulating cerebrovascular compliance in humans are unknown. This retrospective study investigated the impact of neural, endothelial, and myogenic mechanisms on the regulation of vascular compliance in the cerebral vascular bed compared with the forearm vascular bed. An index of vascular compliance (NEW & NOTEWORTHY Vascular compliance represents an important determinant in the dynamics and regulation of blood flow through a vascular bed. However, the mechanisms that regulate vascular compliance remain poorly understood. This study examined the impact of neural, endothelial, and myogenic mechanisms on cerebrovascular compliance compared with forearm vascular compliance. Distinct pharmacological blockade of α-adrenergic, endothelial muscarinic, and myogenic inputs altered cerebrovascular and forearm vascular compliance. These results further our understanding of vascular control and blood flow regulation in the brain. A combination of steady-state and pulsatile flow mechanics is involved in the control of blood flow through a vascular bed . The humIn contrast, the mechanisms governing vascular compliance in the peripheral circulation have been explored including neural and myogenic inputs. For example, sympathoexcitation by lower body negative pressure and a cold pressor test, reduced forearm vascular compliance while elevation of the arm above the heart, changing forearm perfusion pressure and eliciting myogenic responses, produced an increase in forearm vascular compliance . FurtherStudies regarding vascular compliance in the forearm may not translate well to the brain because of the difference in baseline contractile state, neural innervation, and intracranial pressure. Specifically, relative to the forearm, the cerebral vascular bed is chronically dilated, the impact of adrenergic sympathetic neural innervation differs, and the rigid cranium produces a state of elevated extramural pressure for the brain’s circulation. These conditions are expected to influence the stiffness of the cerebral vascular bed relative to the peripheral vascular bed. Therefore, this study aimed to gain new insight to the mechanisms governing cerebrovascular compliance by comparing the mechanisms regulating vascular compliance in the cerebral and forearm vascular beds.2+) channels (involved in the myogenic response) on vascular compliance in healthy humans. We applied a Windkessel modeling approach to calculate cerebrovascular and forearm vascular compliance under control conditions and during distinct drug infusions of phentolamine (nonselective α-adrenergic receptor blockade), glycopyrrolate , and nicardipine (vascular smooth muscle Ca2+ channel blockade). On the basis that vascular contractile state can limit the expression of vascular compliance, the present study tested the hypothesis that sympathetic blockade would increase cerebrovascular compliance because of smooth muscle cell relaxation. On the basis that endothelial muscarinic receptors exert a vasodilatory effect, the present study tested the hypothesis that cholinergic blockade would reduce cerebrovascular compliance because of greater smooth muscle cell activation. On the basis that vascular smooth muscle Ca2+ channels are involved in myogenic responses; the present study tested the hypotheses that their blockade would increase cerebrovascular compliance through smooth muscle cell relaxation. Concurrent measures in the forearm assessed the systemic versus localized cerebral effects of these pharmacological agents.The present study used a pharmacological approach to study the impact of neural, endothelial, and myogenic mechanisms on cerebrovascular compliance. Specifically, we examined the impact of α-adrenergic receptors, endothelial muscarinic receptors, and vascular smooth muscle calcium . Experiment 2 was performed nine healthy adults aged 21–30 yr . Experiment 3 was performed in 16 healthy adults aged 21–30 yr . Before all experiments, participants were asked to abstain from caffeine consumption for at least 12 h, alcohol consumption for at least 24 h, and physical exercise for at least 24 h.2 analyzer, Model 17515, VacuMed, Ventura, CA). All signals were collected and stored for offline analysis with data acquisition systems . A 20-guage catheter was inserted into an antecubital vein for drug infusion.As previously reported –8, partiexperiment 1, a 0.14 μg/kg bolus followed by a 0.014 μg/kg/min infusion of phentolamine adequately blocks α-adrenergic effects on the vasculature and reduces total peripheral resistance  for experiment 1, n = 9 participants  for experiment 2, and n = 14 participants  for experiment 3. A total of three participants were excluded from analysis  as they did not meet signal quality required for the analysis. Analysis was completed on a selection of 10 cardiac cycles during steady-state conditions of both the control and drug infusion periods. The individual BP and corresponding MCAv and BAv waveforms were extracted in an alternating pattern  to capture 4 ± 1 respiratory cycles. Previously, we have shown strong reproducibility between separate baseline selections using 10 cardiac cycles (Ci) and forearm vascular Ci. Previously, using a similar analytical approach, we demonstrated strong reproducibility between baseline conditions separated by 5–10 min (Ri) was calculated for the cerebral and forearm vascular beds as the quotient of mean BP over mean MCAv and BAv, respectively. For each beat extracted and input into the model, additional hemodynamic measures were analyzed, including HR, systolic BP (SBP), diastolic BP (DBP), pulse pressure (PP), systolic MCAv, diastolic MCAv, systolic BAv, and diastolic BAv. For each measure, averages were calculated across the 10 values at baseline. Analysis was completed on icipants  females c cycles . For eacicipants  females Ci and Ri. Post hoc paired t tests were performed to evaluate simple main effects of drug in each vascular bed. Paired t tests evaluated differences in the absolute and percent change in Ci between the cerebral and forearm vascular beds. The agreement of change in Ci with each drug between the cerebral vascular and forearm vascular beds was evaluated by Pearson’s product moment correlations or Spearman’s correlations where noted. Paired t tests evaluated the effect of drug infusions (control vs. drug) on hemodynamic variables and P < 0.05, and data are presented as means (SD).A two-way repeated-measures ANOVA evaluated the effect of drug infusions (control vs. drug) and vascular bed  on r 3.0.10 . Statistexperiment 1  and forearm vascular Ri was 13–24 times greater than cerebrovascular Ri .When considering data from the control conditions  in Ci in both the cerebral and forearm sites  and forearm vascular bed . The absolute increase in Ci during phentolamine infusion was not different between the two vascular beds . Phentolamine infusion increased Ci by 131 ± 135% in the cerebral vascular bed and 93 ± 75% in the forearm vascular bed  or percent change .Phentolamine infusion increased Ri by vascular bed  whereas cerebrovascular Ri remained unchanged . Phentolamine infusion did not affect mean BP .When compared with the control condition, phentolamine differentially affected Ci though its affect differed by vascular bed  and forearm vascular Ci  but the absolute change in Ci between control conditions and glycopyrrolate infusion was greater in the forearm vasculature compared with the cerebral vasculature . However, given the difference in control Ci between the cerebral vascular bed (2.8e−4 ± 1.4e−4 cm/s/mmHg) and the forearm vascular bed , the percent change was not different between the vascular beds . A similar relationship was observed with the percent change in Ci between the cerebral and forearm vascular beds .When compared with the control condition, glycopyrrolate increased Ri . A marked increase in HR was also observed following glycopyrrolate infusion (P = 0.09).Glycopyrrolate infusion did not affect cerebrovascular or forearm vascular infusion . NotablyCi in both vascular beds  and forearm vascular bed . However, the percent increase in Ci with nicardipine was greater in the cerebral vascular bed (88 ± 88%) compared with the forearm vascular bed . This was also true when evaluating the relationship between the absolute change in cerebrovascular Ci and the absolute change in forearm vascular Ci . In addition, as further support for differential effects of nicardipine on cerebrovascular Ci and forearm vascular Ci, post hoc analysis of simple main effects revealed a significant increase in cerebrovascular Ci (Ppost hoc= 0.002) but no change in forearm vascular Ci (Ppost hoc = 0.23) with nicardipine.When compared with the control condition, nicardipine infusion increased Ri .Nicardipine infusion did not alter cerebrovascular or forearm vascular Ci was four- to fivefold greater in the forearm vascular bed than the cerebral vascular bed during control conditions. Second, nonselective α-adrenergic blockade with phentolamine increased Ci similarly in the cerebral and forearm vascular beds. Third, endothelial muscarinic receptor blockade with glycopyrrolate produced similar effects on cerebrovascular Ci and forearm vascular Ci. Fourth, blockade of l-type Ca2+ channels with nicardipine induced a significant increase in cerebrovascular Ci but no difference in forearm vascular Ci. Therefore, these data suggest that cerebrovascular Ci is affected by α-adrenergic, endothelial, and myogenic mechanisms with regionally specific differences of l-type Ca2+ channels.There are four major findings of the present study. First, across all three experiments, A few key factors should be considered when interpreting the findings from this study. The load-bearing function of elastin is greater at low pressures or unstretched vessels, producing highly compliant conditions, whereas collagen exerts proportionately greater support of wall tension with progressive distension leading to a curvilinear relationship between pressure and diameter –17. AlsoCi was four- to fivefold greater than cerebral vascular Ci during control conditions. This may be expected based on several factors. For example, compared with systemic arteries, the cerebral arteries lack an external elastic lamina and exhibit fewer elastic fibers in the tunica media , in addition to endothelial muscarinic receptors (M3). However, the rise in heart rate would be expected to reduce Ci  in the brain’s circulation.The increase in Ri in the present study. This observation was unexpected as nicardipine reduces systemic vascular resistance and BP in hypertensive populations  regulation of vascular resistance between the forearm circulation and cerebral circulation and also differing myogenic regulation of vascular compliance between the two sites. The observation of augmented Data are available on request.This work was supported by National Heart, Lung, and Blood Institute Grant HL-093113; Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant RGPIN-2018-06255; and Canadian Institutes of Health Research (CIHR) Grant 201503MOP-342412-MOV-CEEA (to J.K.S.). M.E.M. was supported by an Ontario Graduate Doctoral Scholarship (O.G.S.). S.A.K. is supported by NSERC Discovery Grants RGPIN-2022-05293 and DGECR-2022-00320.No conflicts of interest, financial or otherwise, are declared by the authors.M.E.M., J.W.H., C.O.T., and J.K.S. conceived and designed research; J.W.H. and C.O.T. performed experiments; M.E.M. and S.A.K. analyzed data; M.E.M., S.A.K., M.Z., J.W.H., C.O.T., and J.K.S. interpreted results of experiments; M.E.M. prepared figures; M.E.M., S.A.K., M.Z., J.W.H., C.O.T., and J.K.S. drafted manuscript; M.E.M., S.A.K., M.Z., J.W.H., C.O.T., and J.K.S. edited and revised manuscript; M.E.M., S.A.K., M.Z., J.W.H., C.O.T., and J.K.S. approved final version of manuscript.


Esophageal squamous cell carcinoma (ESCC) is one of the most common and lethal malignant diseases. Immunotherapy has been widely studied and has exhibited potential in ESCC treatment. However, there are only a portion of ESCC patients have benefited from immunotherapy. We herein identified immunotherapeutic response-related signatures (IRRS) and evaluated their performance in ESCC prognosis and immunotherapeutic responsiveness.We constructed an IRRS using the gene expression data of 274 ESCC patients based on y -30significantly differentially expressed genes, which were compared responders and non-responders from various patient cohorts treated with immunotherapy. Survival analysis was performed in both the GSE53625 and TCGA-ESCC cohorts. We also explored the differences in the tumor microenvironment between the high-IRRS and low-IRRS score groups using single-cell data as a reference. Three immunotherapy cohorts were used to verify the value of the IRRS in predicting immunotherapy response.Twelve immunotherapy-related genes were selected to construct a signature score and were validated as independent prognostic predictors for patients with ESCC. Patients with high IRRS scores exhibited an immunosuppressive phenotype. Therefore, patients with low IRRS scores may benefit from immunotherapy.IRRS score is a biomarker for immunotherapy response and prognosis of ESCC. Esophageal cancer (EC) ranks the 9th most common cancer worldwide and 6th leading cause of cancer deaths globally . EsophagPD‐L1 expression patients  database (https://www.ncbi.nlm.nih.gov/geo/). The GSE53625 dataset comprised 179 ESCC samples and 179 adjacent, matching non-tumor tissues. The single cell RNA (scRNA) sequencing data GSE160269 dataset was composed of 208,659 single cells from 60 ESCC samples.Two ESCC datasets (GSE53625 and GSE160269) were downloaded https://portal.gdc.cancer.gov). The melanoma immunotherapy cohorts (GSE78220 and GSE91061) were also downloaded from the GEO database. Immunotherapy cohort IMvigor210 was downloaded from the IMvigor210CoreBiologies package (https://isb-cgc.shinyapps.io/iatlas/)   . Genes tIRRGs differentially expressed between paracancerous (n = 179) and cancerous (n = 179) tissues were identified using the R limma package(3.50.3)  based onGene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment Analysis (GSEA) analyses were performed using the R cluster Profiler package (4.2.2) . DEGs bescRNA-seq data were obtained from the GSE160269 dataset. We profiled the transcriptomes of 208,659 single cells, including 97,631 immune and 111,028 non-immune cells from 60 ESCC samples. The data provided eight main cell populations, namely epithelial cells , fibroblasts , endothelial cells , pericytes (N = 3102), fibroblastic reticular cells , T cells N = 69,278), B cells , and myeloid cells  . Receiver operating characteristic (ROC) curves were plotted using the R package “timeROC” (0.4). The statistical significance of normally distributed variables was evaluated using unpaired Student’s We downloaded a genelist about immunotherapeutic response from the TISIDB database  and inteUnivariate Cox analysis of the 798 immunotherapeutic response-related genes showed that 54 genes were significantly associated with OS in GSE53625 ESCC cohort.  +  +  +  +  +  +  +  +  +  +  + . Patients in the training cohorts were classified into low- and high-risk groups based on the cut-off value of the risk score  for survival prediction. Based on the optimum λ value, twelve genes were selected to be the IRRS, indicating the prognostic signature  and M2 macrophages markers  were expressed in both intermediate clusters of TAM03  were herein selected to establish the IRRScore prognostic model. These genes have been reported to associated with tumor progression and immune suppressive TME in ESCC and other cancers. Specifically, HPSE, ANO1, and SERPINH1 have been shown to be prognostic indicators of ESCC . There is evidence indicates that exhausted T-cells leading to poor responsiveness to treatment with immune checkpoint blockade . The comOur study still has some limitations. First, the twelve-gene signature was developed and validated based on a public database; thus, more experimental data are needed. Second, there are 58.1% and 55.8% patients have been treated with chemotherapy or radiotherapy in GSE53625 and TCGA-ESCC cohorts. These proportions are similar to patients with prior treatment in immunotherapy cohorts which has been used as validation cohorts, such as 51.5% in GSE91061, 42.8% in GSE78220 and 78.1% in IMvigor210 cohorts. Since we are establishing a model to predict the survival and immunotherapeutic effect of ESCC patients, whose gene expression status can reflect the gene expression of patients before immunotherapy. The current obtainable ESCC and immunotherapy cohort are very limited. So, we can’t find a cohort which had patients without prior treatment. We will use more accurate cohorts for validation in the future when these dataset is obtainable. Third, the number of immunotherapy cohorts with high-throughput sequencing is very limited. We need to verify the applicability of our research results in patients with ESCC receiving immunotherapy. Moreover, the molecular functions and mechanisms of prognostic IRRS should be further elucidated using basic experiments.https://www.ncbi.nlm.nih.gov/geo/ and https://portal.gdc.cancer.gov/. The accession numbers are included in the article.The datasets used in this study can be acquired here: YP and BZ conceived and designed the study. BZ, MZ, JL, and WD analyzed the bioinformatics data. BZ, PZ and YP wrote the manuscript. All authors contributed to the article and approved the submitted version.