Campylobacter jejuni infections inducing campylobacteriosis including post-infectious sequelae such as Guillain-Barré syndrome and reactive arthritis are rising worldwide and progress into a global burden of high socioeconomic impact. Intestinal immunopathology underlying campylobacteriosis is a classical response of the innate immune system characterized by the accumulation of neutrophils and macrophages which cause tissue destruction, barrier defects and malabsorption leading to bloody diarrhea. Clinical studies revealed that enteritis and post-infectious morbidities of human C. jejuni infections are strongly dependent on the structure of pathogenic lipooligosaccharides (LOS) triggering the innate immune system via Toll-like-receptor (TLR)-4 signaling. Compared to humans, mice display an approximately 10,000 times weaker TLR-4 response and a pronounced colonization resistance (CR) against C. jejuni maintained by the murine gut microbiota. In consequence, investigations of campylobacteriosis have been hampered by the lack of experimental animal models. We here summarize recent progress made in the development of murine C. jejuni infection models that are based on the abolishment of CR by modulating the murine gut microbiota and by sensitization of mice to LOS. These advances support the major role of LOS driven innate immunity in pathogenesis of campylobacteriosis including post-infectious autoimmune diseases and promote the preclinical evaluation of novel pharmaceutical strategies for prophylaxis and treatment.Human  Campylobacter jejuni is recognized as one of the leading causes of infectious bacterial enteric infections worldwide [Campylobacter species and subspecies, C. jejuni and C. coli are the most common subpopulations causing human infections [The foodborne bacterial pathogen orldwide ,4,5,6,7.orldwide . Among mfections .C. jejuni bacteria are highly motile and grow under microaerophilic conditions in a temperature range between 37 and 42 °C [C. jejuni is present in surface waters and forms part of the natural intestinal microbiota of a wide range of wild animals as well as of agriculturally essential mammals and birds, especially poultry [C. jejuni between the two species, underlining the significance of poultry as a source of human C. jejuni infections [C. jejuni [The Gram-negative, slender, spirally-curved, flagellated nd 42 °C ,11,12. C poultry ,11. Recefections . In fact. jejuni ,15.Campylobacter strain and the host immune status, patients might present with a highly acute and severe symptom complex varying from watery diarrhea without fever and/or abdominal cramps to severe campylobacteriosis characterized by purulent bloody inflammatory diarrhea and systemic inflammatory responses including fever [C. jejuni [C. jejuni LOS [C. jejuni LOS in intestinal immunopathogenesis of campylobacteriosis, which will be discussed further. Depending on the ng fever ,16,17,18ng fever ,20. Howeng fever ,22,23. Ajuni LOS ,25, the C. jejuni enters the gut via ingestion of food contaminated with a low number of live bacteria [C. jejuni pass the barriers of the viscous mucus layer  following broad-spectrum antibiotic treatment, and also upon re-association with human as opposed to murine gut microbiota by fecal microbiota transplantation, these mice can be effectively colonized by the pathogen upon peroral challenge and display typical histopathologic pro-inflammatory features of campylobacteriosis in their intestines [In contrast to humans, conventional mice are well protected from nization . This canization   were analyzed via mass spectrometry and revealed that almost all strains expressed LOS with structural similarities to gangliosides in nerve fibers [C. jejuni strains [C. jejuni strains with sialylated LOS have a higher invasive potential in cell cultures, and that inactivation of LOS sialyltransferase results in the loss of such invasiveness [C. jejuni invasiveness, given that only 23% of isolates from patients with C. jejuni bacteremia belonged to LOS locus classes indicative of a potential to sialylate the LOS [The results from several clinical studies demonstrate that the hyper-activation of the innate immune system by sialylated h as GBS ,72  is genetically similar to the completely sequenced C. jejuni NCTC 11168 strain, whereas the expressions of LOS genes were shown to strongly vary between respective strains. The presence of the identical LOS locus in both C. jejuni ATCC 43446 and GB11 strains is probably the result of horizontal gene transfer [C. jejuni strains leads to variations in LOS synthesis genes, which might be indeed related to C. jejuni-associated immunopathologies.Molecular analyses demonstrated that the transfer . Additiotransfer . These fC. jejuni waaF gene  results in the truncation of the LOS inner core [p-coumaric acid, epigallocatechin gallate, and hesperidin [C. jejuni ΔlgtF, ΔwaaF, and parental strains revealed that LOS may be involved in bacterial colonization properties [C. jejuni mutant strains showed that the growth of both mutants was unaffected, indicating that the observed colonization defects of the ΔlgtF and ΔwaaF mutant strains might indeed constitute specific and interesting in vivo phenomena [Interestingly, surface carbohydrate structures such as LOS play essential roles as permeability barriers against hydrophobic antibiotics  and phenner core , causingsperidin . Furtheroperties . Howeverhenomena .C. jejuni across intestinal epithelial barriers [C. jejuni strains  resulted in less distinct invasiveness into human epithelial cells as compared to the respective wildtype bacteria [C. jejuni isolates expressing LOS mimicking ganglioside structure were shown to strongly attach to the Caco-2 intestinal epithelial cells, as opposed to C. jejuni control isolates [C. jejuni ganglioside-like LOS-expressing isolates are linked to severe enterocolitis with bloody diarrhea [As mentioned above, LOS is essential for both cellular invasion and translocation of barriers ,40,41,78bacteria . In addiC. jejuni is a strategy to evade recognition by the host immune system. This mechanism is common to bacterial pathogens in general.Finally, the variation of the LOS structure that may even mimic human ganglioside-like structures by C. jejuni LOS by specific binding of the lipid A moiety to the TLR-4 receptor complex of the innate immune system  exerted by the murine gut microbiota composition [Several animal models, such as a larvae , colostra larvae , gnotobia larvae , naïve sa larvae , and fera larvae  were sucnfection . The useposition ,46,50. C. jejuni is able to colonize the intestines of germfree BALB/c and C57BL/6 mice [C. jejuni are able to colonize the gastrointestinal tract of secondary abiotic wildtype mice and secondary abiotic mice that had been associated with a complex human gut microbiota following FMT, resulting in pro-inflammatory immune responses in the colon upon peroral infection [C. jejuni is abrogated by modifying the microbiota composition towards increased intestinal commensal Escherichia coli loads, as seen following E. coli feeding via the drinking water, during intestinal inflammation or in infant mice immediately after weaning [C. jejuni was even able to colonize and cause clinical signs such as severe diarrhea with increased mucus discharge, reduced weight gain, and sometimes bloody stool, as early as 5 days post-infection [C. jejuni LOS are not developed in the early stage of murine life, as is discussed further below (Chapter 9).On the other hand, it has been known for decades that L/6 mice ,59,60. Tnfection ,46,107.  weaning ,108. In nfection . These fC. jejuni infection model through application of a single antibiotic compound to mice [Bacteroidetes and Clostridia, and conversely, a promoted growth of Lactobacilli within the murine intestinal lumen [C. jejuni to colonize the intestinal lumen at high loads. Based on the finding that upon C. jejuni infection vancomycin treated mice that were deficient in Single Ig IL-1-related receptor (SIGIRR) develop intestinal inflammation, the authors proposed Sigirr−/− mice as a novel campylobacteriosis model. Following peroral C. jejuni infection, Sigirr−/− mice exerted more severe gross pathology as compared to wildtype mice, which was accompanied by higher expression levels of pro-inflammatory cytokines such as TNF-α, IFN-γ, and IL-17 in the former versus the latter [C. jejuni induced inflammatory disease [C. jejuni LOS. Stahl et al. developed an in vivo  to mice . Peroralal lumen ,110. Thee latter . Further disease ,111. In  disease  and, thuC. jejuni colonize the intestinal tract of five to six months old IL-10−/− mice with murine intestinal microbiota suffering from chronic colitis; however, the clinical signs for severe human campylobacteriosis such as bloody diarrhea were missing [−/− mice as a clinical model for campylobacteriosis developed by Mansfield et al. [−/− mice were subjected to broad-spectrum antibiotic treatment starting immediately after weaning in order to deplete the commensal gut microbiota and, hence, to abrogate physiological CR preventing C. jejuni infection and furthermore, in order to eliminate potential colitogenic stimuli from the commensal gut microbiota, leading to chronic colitis in IL10−/− mice after 3 months or more [C. jejuni infection, these so-called secondary abiotic IL10−/− mice were stably colonized with high pathogenic loads and suffered from non-self-limiting acute enterocolitis, characterized by wasting and bloody inflammatory diarrhea, thus mimicking key features of severe human campylobacteriosis [C. jejuni-induced innate and adaptive pro-inflammatory immune responses were not limited to the intestinal tract but could also be observed in extra-intestinal including systemic compartments. The isolation of C. jejuni from mesenteric lymph nodes (MLNs), liver, kidneys, spleen and cardiac blood indicated that invading bacteria had been able to translocate from the inflamed intestines to extra-intestinal and even systemic compartments, further perpetuating the fatal inflammatory scenario. Overall, the secondary abiotic IL-10−/− mouse model has been proven valuable to unravelling the immunopathological impact of distinct C. jejuni virulence factor involved in human disease [Similar to SIGIRR deficiency, the lack of the anti-inflammatory cytokine IL-10, which inhibits TLR-dependent innate immune responses, abolished the murine resistance to LOS and LPS ,115,116. missing ,61. Inspd et al.  and Lippd et al. , our gro or more ,48,50. W disease ,48,52,64−/− IL-10−/−) mice were far less compromised and exhibited less distinct intestinal as well as extra-intestinal including systemic sequelae upon C. jejuni infection. These results provide further evidence for a major role of TLR-4 mediated, LOS dependent immunopathological mechanisms underlying campylobacteriosis [C. jejuni infection experiments in TLR-4 deficient Sigirr−/− mice revealed similar results [−/−Sigirr−/− mice, C. jejuni colonized the intestines at high levels, but induced rather mild, if any, signs of enteritis. Compared to Sigirr−/− counterparts, TLR-4 deficient Sigirr−/− animals displayed less TNF-α and IFN-γ expression in ceca upon infection, which was comparable to that assessed in uninfected control mice [Remarkably, TLR-4 and IL10 double deficient . In summary, the abolishment of CR against  disease . Additio in mice  and that in mice . The extC. jejuni-induced host responses and virulence factors in genetically modified mice with or without a specifically modified gut microbiota. As mentioned previously, it has been shown that the murine microbiota is an essential physiological prerequisite for an effective CR directed against C. jejuni. Besides secondary abiotic mice, mice harboring a human gut microbiota, infant mice and mice displaying elevated commensal intestinal E. coli loads constitute valuable tools for investigating C. jejuni-host interactions and infection-induced immunopathological responses of varying severities [Many recent studies investigated various immunopathological aspects of campylobacteriosis, including verities ,48,131.C. jejuni. Most importantly, upon C. jejuni infection those infant mice display the typical course of self-limiting campylobacteriosis seen in humans [Given the central role of IL-10 in maintaining gut homeostasis by the suppression of inflammatory responses ,133, it n humans . MoreoveC. jejuni [E. coli lacking any invasive or other pathogenic properties do not induce pathology in this model [C. jejuni infection by the potent immune suppressor sirolimus (rapamycin), which inhibits intracellular signaling by mammalian target of rapamycin (mTOR). The mTOR complexes 1 and mTORC2 play important roles in regulating the activation of innate immune cell populations such as macrophages and dendritic cells and induce cytokine production [C. jejuni-induced colitis independently of T cell activation by recruitment of neutrophilic granulocytes to the infected intestines and subsequent massive TNF secretion [C. jejuni-induced neutrophil accumulation which correlated with reduced inflammatory responses. In fact, neutrophils have been shown to exert potent pro-inflammatory responses upon LPS stimulation in vitro characterized by TLR-4 dependent TNF-α and IL-6 secretion [C. jejuni has been shown to produce a cytolethal distending toxin, a genotoxin with DNase activity that is supposed to be involved in the pathogenesis of colorectal cancer [C. jejuni-induced carcinogenesis [C. jejuni-induced colitis or colorectal tumorigenesis can be attenuated by inactivation of mTOR signaling using pharmacological interventions such as rapamycin or PI3K-γ inhibitors [In particular, the LOS sensitized secondary abiotic IL-10 deficient mice were successfully used for groundbreaking studies for a better molecular understanding of immunopathogenesis of campylobacteriosis. In this model, murine symptoms mimic human disease and induction of intestinal inflammation strictly depends on the motility and invasive properties of . jejuni . It is ois model . This muoduction ,135. Theoduction ,137. Addoduction . In addiecretion ,138. By ecretion ,138. To ecretion . Upon LPecretion . Moreovel cancer ,140,141.ogenesis . Hence, hibitors ,139. The−/− (NOD2−/− IL-10−/−) mice were used to unravel the role of the innate immune receptor nucleotide-oligonucleotide-domain 2 (NOD2) during campylobacteriosis and clearance of C. jejuni infection [C. jejuni infection, secondary abiotic NOD2−/− IL-10−/− mice harbored the pathogen at high loads, but developed less severe enterocolitis as compared to IL-10−/− counterparts [Campylobacter induced GBS [In the past few years, secondary abiotic as well as conventional NOD2 deficient IL-10nfection ,145,146.terparts , indicatterparts . These iuced GBS ,148,149.Even though the exact roles of NOD2 during campylobacteriosis remain unknown, several reports have emphasized the complex modulatory crosstalk between NOD2 and TLR-4 signaling especially in pro-inflammatory cytokine regulation ,153,154,C. jejuni-induced inflammation in the course of campylobacteriosis [Most recently, the secondary abiotic IL10 deficient murine model of campylobacteriosis was standardized and could be developed to the preclinical level for pharmaceutical analysis of alternative drugs, including ascorbate and vitamin D, which effectively suppressed teriosis ,160.C. jejuni in both humans and mice hampers the development of vaccines against C. jejuni. This is mainly due to the fact that it is much more difficult to develop effective vaccines against endotoxin-driven morbidities as compared to exotoxin-mediated diseases [C. jejuni-LOS and human gangliosides, antibodies directed against LOS might react with human peripheral nerves [C. jejuni LOS antibodies with GBS constitutes a substantial challenge if one might consider LOS a potential target molecule for vaccine development. Likewise, the production of antibodies directed against C. jejuni flagella could be difficult since the corresponding structural proteins are heavily glycosylated and show massive structural variations among bacterial strains [C. jejuni. The advantage, as opposed to LOS, is that neither mimicry between gangliosidic surface structures and C. jejuni CPS nor association of CPS with GBS have been described to date [kpsS mutant) has been shown to increase the susceptibility of C. jejuni to antibiotic and phenolic compounds with antibacterial properties [kpsS gene encodes a β-linked 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) transferase playing an important role in CPS synthesis [C. jejuni kps mutant strain was unable to colonize the murine intestines as opposed to the wildtype counterpart [C. jejuni has provided 100% protection against diarrheal disease in monkeys [C. jejuni so far [The major role of the endotoxin LOS in the inflammatory response to diseases . In addil nerves ,74. Ther strains . However to date . Additiooperties . The kpsynthesis . Moreoventerpart . Additio monkeys . Howeveri so far , the devCampylobacter infections result in severe inflammation of the intestinal mucosa, whereas colonized avian species harbor C. jejuni as intestinal commensal bacteria and are usually lacking any clinical signs [C. jejuni infection rates in humans, and in consequence, the prevalence of severe human campylobacteriosis with the risk of subsequent post-infectious sequelae, the vaccination of broiler chickens against C. jejuni is proposed to be one of the most efficient strategies to reduce the intestinal C. jejuni loads in chickens and, thus, the risk of transfer to humans via consumption of contaminated meat. The comparison of the C. jejuni colonization properties in humans and chickens revealed host-specific differences in susceptibility to the bacteria. For example, the characterization of chicken TLRs showed major species-specific characteristics [C. jejuni induces IFN-β, a key mediator of systemic inflammation, through TLR-4 activation in humans, but not in chicken [C. jejuni is highly susceptible to the chicken host defense peptide cathelicidin-2 exerting antimicrobial activities [Campylobacter is able to colonize the avian gastrointestinal tract (with up to 109 CFU/g feces) without causing overt clinical signs [C. jejuni-induced immune responses in humans and chicken are very different, applying vaccines against C. jejuni to chickens could only reduce the Campylobacter transfer to humans. However, there are several studies using clinical strains such as C. jejuni 81-176 in chicken for vaccine development strategies [Human al signs . In ordeeristics . C. jeju chicken . Furthertivities . These ral signs ,167,168.rategies ,169. C. jejuni 81-176 strain [C. jejuni loads by up to 4.2 log orders of magnitude in one trial. However, these promising results could not be reproduced, despite a strong induced immune response upon application of the same antigen [C. jejuni N-glycan as a potential antigenic structure for vaccine development, since it constitutes a surface carbohydrate with immunogenic properties in rabbits and humans [C. jejuni 81-176 strain in SPF Leghorn chickens [Results from an in vivo study with Ross broiler chickens revealed four promising antigens  as potential vaccine candidates against the 6 strain . The ide antigen . In addid humans ,171. In chickens . C. jejuni, as compared to mice and humans [C. jejuni mainly locate in the mucous layer of the crypts in chickens and do not directly adhere to the avian intestinal epithelial cells [C. jejuni rarely invade the intestinal epithelium and if the internal organs are affected, no clinical signs of disease are observed in colonized chickens [E. coli-LPS  into chickens induced only rather mild clinical signs such as diarrhea and gasping, and the chickens recovered within two days post application. However, a LPS dose of 45.5 mg per kg body weight was lethal to mice within four hours [C. jejuni-LOS) have been shown to activate pro-inflammatory immune cytokine responses [Salmonella minnesota endotoxin to humans induced an inflammatory response with septic shock-like symptoms that continued for several days [Finally, it is noteworthy that chickens are more resistant to endotoxin derived from Gram-negative bacteria, such as d humans ,172. In al cells ,174,175.chickens ,175,176.esponses . In a clral days . C. jejuni.These findings further underline that differences in LOS susceptibility and resistance between vertebrate species such as humans on one hand and chicken and mice on the other might provide explanations for the completely divergent species-specific reactions to C. jejuni LOS as a main pathogenicity factor in human campylobacteriosis and the extreme LOS resistance of mice and birds has provided a substantial key to the understanding of symptomless C. jejuni colonization in chickens on one hand, versus severe infection in humans on the other. In line with a better appreciation of the CR created by the individual microbiota of vertebrates, these historically neglected but valid insights enabled the development of several novel murine models for campylobacteriosis research—all based on the reduction in species-specific resistance to both, bacterial colonization and LOS resistance. The resulting LOS sensitized murine C. jejuni infection models lacking CR mimic key features of human campylobacteriosis and will help to unravel the molecular mechanisms underlying the immunopathology of intestinal infection as well as post-infectious sequelae. Their standardization and use for preclinical pharmaceutical evaluation have paved the way for the identification of novel therapeutic and prophylactic options in order to combat campylobacteriosis, including collateral damages, in the future. The major role of innate immunity and


Prunus davidiana (Rosaceae) is of great importance horticulturally as the rootstock of some fruit trees. Here, the complete chloroplast genome of P. davidiana was assembled based on the Illumina reads. The complete cp genome of P. davidiana was 157,660 bp in length and contained a pair of inverted repeat  regions, which were separated by the small single-copy  and the large single-copy  regions. It encoded 135 genes. The phylogenetic analysis revealed that P. davidiana is sister to other peaches. This result will be useful for future studies of the lineage. Prunus davidiana (Carriére) de Vos ex L. Henry (Rosaceae) is always used as the rootstock of fruit trees such as peach, plum in China, and also as an ornamental plant with attractive pink flowers, showing a great horticultural value . A voucher specimen (LMR20160501) was deposited in the Herbarium of Northwest A&F University (WUK), China. Total genomic DNA was extracted with the modified cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle https://github.com/quxiaojian/PGA). The genome map was generated in the webserver OGDRAW (http://ogdraw.mpimp-golm.mpg.de)  regions, which were separated by a small single-copy  region and a large single-copy  region. The whole cp genome encoded 135 genes including 88 protein-coding genes (PCG), 39 tRNA genes, 8 rRNA genes. Of these genes, 16 genes  had one intron and 2 genes (clpP and ycf3) had two introns. Most genes occurred in a single copy, while six PCGs , seven tRNA genes , and four rRNA genes  in IR regions are duplicated. The overall GC content of P. davidiana cp genome is 36.8% with 34.6, 30.3, and 42.6% GC in the LSC, SSC, and IR regions, respectively.The complete cp genome of Prunus L. were used to obtain the phylogenetic position of P. davidiana. Prinsepia utilis and Prinsepia sinensis were used as the outgroup. All the cp genome sequences were aligned in MAFFT (Katoh and Standley P. davidiana and other species of subgenus Amygdalus form a small clade (Prunus based on several molecular markers (Bortiri et al. P. davidiana will be valuable for future horticultural studies.Totally 12 additional cp genomes of the genus ll clade , which i


Monochamus saltuarius (Gebler) is the newly discovered insect vector of the pine wood nematode (Bursaphelenchus xylophilus) in China. Adult M. saltuarius prefers to oviposit on the host plant Pinus koraiensis, rather than P. tabuliformis. However, the genetic basis of adaptation of the larvae of M. saltuarius with weaken dispersal ability to host environments selected by the adult is not well understood.In many insect species, the larvae/nymphs are unable to disperse far from the oviposition site selected by adults. The Sakhalin pine sawyer M. saltuarius larvae, i.e., P. koraiensis and P. tabuliformis were investigated. Compared with P. koraiensis, P. tabuliformis had a substantially higher content of various free amino acids, while the opposite trend was detected for fatty acid content. The transcriptional profiles of larval populations feeding on P. koraiensis and P. tabuliformis were compared using PacBio Sequel II sequencing combined with Illumina sequencing. The results showed that genes relating to digestion, fatty acid synthesis, detoxification, oxidation-reduction, and stress response, as well as nutrients and energy sensing ability, were differentially expressed, possibly reflecting adaptive changes of M. saltuarius in response to different host diets. Additionally, genes coding for cuticle structure were differentially expressed, indicating that cuticle may be a potential target for plant defense. Differential regulation of genes related to the antibacterial and immune response were also observed, suggesting that larvae of M. saltuarius may have evolved adaptations to cope with bacterial challenges in their host environments.In this study, the free amino and fatty acid composition and content of the host plants of M. saltuarius relating to host plant adaptation. Results from this study help to illustrate the fundamental relationship between transcriptional plasticity and adaptation mechanisms of insect herbivores to host plants.The present study provides comprehensive transcriptome resource of The online version contains supplementary material available at 10.1186/s12864-021-07498-1. For insect herbivores, adaptation to host plants is crucial to their ability to colonize a variety of environments . Host plBursaphelenchus xylophilus) is a plant parasitic nematode and major cause of pine wilt disease in Asia and Europe  [The real-time quantitative PCR (qRT-PCR) assay was conducted to validate the results of our transcriptome sequencing analysis. Reverse transcription was performed using the PrimeScript first strand cDNA synthesis kit  according to the manufacturer’s protocol. The gene-specific primers were designed using Primer5 . The  Ctnom)] .Additional file 1: Figure S1. Read length of CCS and FLNC. a CCS. b FLNC.Additional file 2: Figure S2. The completeness of transcripts assessed by benchmarking universal single-copy ortholog (BUSCO). The x-axis represents the percentage of detected BUSCOs. The light blue diamond represents the complete (C) and single-copy (S) genes; the dark blue represents complete and duplicated (D) genes; the yellow diamond represents fragmented (F) genes; the red diamond represents the missing (M) genes. Total number of core genes queried was 1066.Additional file 3: Table S1. Annotation of the 32,304 non-redundant transcripts.Additional file 4: Figure S3. Species distribution of the top BLAST hits of the total homologous sequences.Additional file 5: Figure S4. Gene ontology classification of non-redundant transcripts. The 13,144 transcripts were classified into three functional categories: molecular function, biological process and cellular component.Additional file 6: Figure S5. KEGG pathway distributions of non-redundant transcripts. The genes according to KEGG metabolic pathway involved was divided into six branches: Metabolism, Genetic information processing, Environmental information processing, Cellular processes, Organismal systems, and Human disease.Additional file 7: Figure S6. Number of transcript factors identified in the present study.Additional file 8: Table S2. Summary of SSRs identified in the transcriptome of Monochamus saltuarius.Additional file 9: Table S3. Annotation of the 2166 differentially expressed genes.Additional file 10: Figure S7. Cluster analysis of differentially expressed genes. Different colors indicate different levels of gene expression. The firebrick color indicates upregulated expression, whereas the navy color indicates downregulated expression. Pk: the larvae feeding on Pinus koraiensis; Pt: the larvae feeding on P. tabuliformis.Additional file 11: Table S4. List of KEGG pathways with P-value < 0.05.Additional file 12: Figure S8. GO enrichment of differentially expressed genes. The 20 most enriched GO terms are shown together with their –log10 and number of genes.Additional file 13: Table S5. Primers used in qRT-PCR.


Koumiss is a naturally fermented mare’s milk. Over recent decades, numerous studies have revealed the diversity of lactic acid bacteria in koumiss. However, there is limited information available regarding its secondary major component yeast profile.Lactobacillus helveticus, Lactobacillus kefiranofaciens, Lactococcus lactis, Lactococcus raffinolactis, and Citrobacter freundii. The main yeast species were Dekkera anomala, Kazachstania unispora, Meyerozyma caribbica, Pichia sp.BZ159, Kluyveromyces marxianus, and uncultured Guehomyces. The bacterial and yeast Shannon diversity of the Xilinhaote-urban group were higher than those of the Xilingol-rural group. The most dominant organic acids were lactic, acetic, tartaric, and malic acids. Lactic acid bacteria species were mostly responsible for the accumulation of those organic acids, although Kazachstania unispora, Dekkera anomala, and Meyerozyma caribbica may also have contributed. Redundancy analysis suggested that both bacteria and yeast respond to koumiss flavor, such as Lactobacillus helveticus and Dekkera anomala are associated with sourness, astringency, bitterness, and aftertaste, whereas Lactococcus lactis and Kazachstania unispora are associated with umami.A total of 119 bacterial and 36 yeast species were identified among the 14 koumiss samples. The dominant bacterial species in koumiss were Our results suggest that differences were observed in koumiss microbiota of Xilinhaote-urban and Xilingol-rural samples. The biodiversity of the former was higher than the latter group. Positive or negative correlations between bacteria and yeast species and taste also were found. Koumiss is a naturally fermented mare’s milk with a long history of consumption in the Mongolian steppes. Its name is derived from the Kumanese, who survived as a Kumane River tribe on the central Asian steppes until 1235. When fully renneted, the proteins in horse milk form no visible curd . The LacThe flavor of dairy products is directly and indirectly derived from factors such as the raw milk quality, production process, and activities of diverse microbial populations . In partDuring milk fermentation, the concentrations of some organic acids (lactic and acetic acid) increase, while those of other organic acids  decrease . Short-cChemical composition analysis provides quantitative data that are not instructive in terms of analysis of taste; therefore, we employed a taste-sensing system with advanced taste sensors that were developed based on correlations with human sensory scores, and are applied in assessments of milk and beverage products –20. Tradp = 0.068), whereas the yeast Shannon diversity index was higher in Xilinhaote-urban than Xilingol-rural samples (p = 0.001) ; it had a lower abundance in Xilingol-rural samples than Xilinhaote-urban samples (p = 0.012). There were no significant differences in dominant yeast species abundance.Sequencing of the full-length 16S rRNA gene using the SMRT technology revealed high taxonomical resolution of the Xilinhaote-urban and Xilingol-rural groups at the species level  Fig. . The mosKazachstania unispora (K. unispora) is Saccharomyces unisporus. Yeast species with fluctuations in abundance of < 5% include Meyerozyma caribbica (M. caribbica), Pichia sp.BZ159 (P. BZ159), and uncultured Guehomyces.A total of 119 bacterial and 36 yeast species were identified among the 14 koumiss samples. The bacterial and yeast species with a relative abundance of > 1% are summarized Tables  and presp < 0.05).The organic acids detected in koumiss included oxalic, tartaric, malic, lactic, acetic, citric, and succinic acid, with an average content of 6.54 g/L. The most dominant acids across all samples were lactic , acetic , tartaric , and malic  acids  was positively correlated with that of Dekkera anomala  and P. BZ159 (p < 0.01), and negatively correlated to that of K. unispora (p < 0.05). The abundance of the second most dominant species, L. lactis, was positively correlated with that of K. unispora (p < 0.01) and negatively correlated with that of D. anomala and uncultured Guehomyces (both p < 0.05).In addition to the associations of bacterial and yeast species compositions, a correlation between the abundance of yeast species and LAB was also observed Fig. . The abuLactococcus raffinolactis (L. raffinolactis), L. lactis, and K. unispora, and negatively correlated with that of Lactobacillus kefiranofaciens (L. kefiranofaciens), D. anomala, and uncultured Guehomyces. Interestingly, the abundance of L. raffinolactis and L. lactis was positively correlated with acetic acid accumulation. The abundance of L. kefiranofaciens and M. caribbica was positively correlated with citric acid accumulation, and negatively correlated with tartaric and malic acid accumulation. The abundance of L. kefiranofaciens and unculturedGuehomyces was positively correlated with that of succinic acid, whereas L. lactis abundance had a negative correlation.A complex correlation between koumiss microbiota and organic acid accumulation was observed Fig. . Lactic p = 0.06) and aftertaste-A  than Xilinhaote-urban samples  was positively correlated with sweetness (p < 0.01).Saltiness, umami, and sweetness were clustered together, and were positively correlated with tes Fig. . Only KlL. helveticus and D. anomala were strongly associated with sourness, astringency, bitterness, and aftertaste. L. lactis and K. unispora were strongly associated with umami and lactic and acetic acids. K. marxianus was associated with malic, tartaric, and oxalic acids. L. kefiranofaciens was associated with citric acid.Redundancy analysis (RDA) was performed to identify the species in koumiss associated with the various types of tastes and organic acids Fig. . L. helvKoumiss taste can be classified into three types according to the lactic acid content: strong (0.91–1.08%), moderate (0.73–0.90%), and weak (0.54–0.72%) . Accordi, Citrobacter freundii that had not been reported previously; the dominant yeast species were M. caribbica and uncultured Guehomyces. One observation in our study that was inconsistent with previous research was that the abundance of Enterobacter genus was < 1%; this may be because the samples were freshly fermented koumiss, whereas Enterobacter was found to be enriched during later fermentation and is related to flavor ripening [Acetobacter genus was lower (1.5 ± 2.13%), and mainly consisted of Acetobacter malorum, Acetobacter pasteurianus, and Acetobacter orientalis. The heterofermentative species included a small amount of Lactobacillus kefiri (0.82 ± 1.86%) and Leuconostoc mesenteroides (0.53 ± 1.45%).Many studies on koumiss bacterial or yeast diversity used culture- or molecular biology-based methods; however, results varied significantly due to differences in the analysis techniques applied, research approaches, and geographic regions –26. In tripening . In our L. lactis was absent from Xilingol-rural koumiss samples, there may be a competition relationship between L. lactis and L. helveticus, because L. helveticus utilizes galactose moieties and glucose, whereas L. lactis only utilizes the latter [Lactococcus lactis ssp. lactis and L. raffinolactis stimulated more acid production during skim milk fermentation [L. raffinolactis strains are able to ferment α-galactosides [L. lactis strains. However, in the current study, although L. lactis and L. raffinolactis showed a trend toward a positive correlation in their abundances, it was not statistically significant. In contrast, the abundances of L. kefiranofaciens and L. raffinolactis were positively correlated. L. kefiranofaciens is rarely isolated from koumiss [Citrobacter freundii (C. freundii) is a common component of the gut microbiome of healthy humans, and of soil, and is a widely distributed opportunistic pathogen [C. freundii was found in one sample at an abundance of 11.37% but was present in very small amounts in other samples; thus, the high-abundance sample may have been due to contamination.Regarding most diverse bacterial composition between two groups, the mesophilic bacteria e latter . Regardientation . One posctosides , which a koumiss  but is a koumiss . Citrobapathogen . C. freuD. anomala and K. marxianus, while the galactose-fermenting yeasts included K. unispora and M. caribbica.Yeasts producing koumiss  can ferment lactose or galactose. In the current study, the lactose-fermenting yeasts were D. anomala, belongs to the genera Dekkera (Brettanomyces), which is known for its ability to spoil alcoholic beverages by producing acetic acid and unpleasant flavors and odors [D. anomala is the teleomorph of Brettanomyces anomalus; it differs morphologically and physiologically from the other species in the genus Dekkera in terms of the formation of blastese and the ability to ferment lactose [D. anomala IGC 5153 exhibited limited ability to use weak acids as its only source of carbon and energy. The acetate carrier was not only inducible, but also subject to glucose repression, similar to the carriers identified in acetic acid–grown cells of yeasts such as S. cerevisiae. D. anomala grew in acetic acid-containing medium with a relatively high total acid content, and over a broad pH range including pH 3.5. D. anomala isolates were obtained from Inner Mongolia Koumiss samples and shown by polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) to be present in an amount of 7.29% [D. anomala was isolated from kefir, koumiss, and Shubat [D. anomala. Because wood is an ecological niche for this strongly fermented species, its presence is likely attributable to the wooden tables used for dry salting the cheese blocks [D. anomala were fermented in crock using a traditional method. This suggests that non-wood containers and stirrers could be used to restrain the overgrowth of this species. In our koumiss samples, D. anomala may have partially contributed to the production of acetic acid, but its correlation with the acetic acid content was negative and nonsignificant  are capable of citrate utilization because they possess a plasmid-encoded citrate transporter gene [L. lactis. In the yeast community, acetic acid-producing species include D. anomala and K. unisporus.For the first time, we systematically identified the organic acids responsible for the sour taste of koumiss. The organic acid composition did not directly reflect the grouping of samples; in fact, it better-reflected the fermentation process. Most organic acids produced by microbes can be divided into two main groups depending on their metabolic origin and the main metabolite sequence of the aerobic organism: those associated with the tricarboxylic acid cycle (TCA) and glycolysis arising from the oxidation of glucose; and those using glucose to produce organic acids. Citric, lactic, and malic acids fall into the first group, whereas acetic acid should be considered a biotransformation of ethanol. Succinic acid is mostly produced using chemical methods. entation , where tbacteria . More imter gene . Our samThere were significant lower content of citric acid in Xilingol-rural than Xilinhaote-urban groups. Citric acid is a common constituent of milk; its abundance in mare’s milk is < 2.3 g/kg. Moreover, it is a substrate for certain LAB, which use it to form aromatic substances such as acetoin and diacetyl , 52. In Guehomyces and L. kefiranofaciens.Succinic acid is a good indicator of yeast inoculation status. It is the main organic acid produced by yeast and is formed in the glyoxylate cycle via oxidation of isocitrate, as well as in the reductive citric acid cycle . In the L. helveticus and D. anomala should be reduced, or those of L. lactis and K. unispora increased, in the koumiss starter culture.In the past, more attention was paid to bacterial as opposed to yeast species, even though both bacteria and yeast contribute to unique koumiss fermentation. The presence of yeast in dairy products contributes to flavor formation via the synthesis of a variety of chemical compounds . The kouL. lactis and yeast found that L. lactis produced acetic acid when co-cultured with some yeasts; moreover, the growth of L. lactis was enhanced in most co-cultures in UHT milk [Studies of the interaction between bacteria and yeast in fermented foods yield inconsistent results due to differences in strains, substrates and food matrices , 57, 58.UHT milk . The preL. helveticus, L. kefiranofaciens, L. lactis, L. raffinolactis, D. anomala, K. unispora, M. caribbica, P. BZ159, K. marxianus and uncultured Guehomyces. The abundance of L. lactis significantly lower in Xilingol-rural samples than Xilinhaote-urban samples. Lactic, acetic, tartaric, and malic acids were the main organic acids that accumulated during koumiss fermentation. LAB species were mostly responsible for the accumulation of those organic acids, although K. unispora, D. anomala, and M. caribbica may also have contributed. The RDA showed that L. helveticus and D. anomala are associated with sourness, astringency, bitterness, and aftertaste, whereas L. lactis and K. unispora are associated with umami. The interaction between bacteria and yeast could be demonstrated more convincingly an orthogonal single-strain fermentation experiment combined with a meta-transcriptome study. Our study provides reference values to improve understanding of the interaction between bacteria and yeast in koumiss. These results could inform the selection of multiple-strain cultures for use as bacteria and yeast mixed starter cultures.The dominant microbes in the weak form of koumiss derived from fermented mare’s milk (in the Xilingol region) analyzed in this study includes Fourteen fresh naturally fermented koumiss samples were collected from the Xilingol region of Inner Mongolia and stored at − 80 °C until analysis. The samples were classified into two groups according to the collection site. “Xilinhaote-urban” samples were collected around Xilinhaote city, where koumiss is produced at the ranch scale , whereas “Xilingol-rural” samples were collected from herdsmen located 100–150 km from Xilinhaote city . We evaluated the koumiss quality and sensory by two senior dairy researchers in situ when samples were collected.Genomic DNA was extracted using an Omega DNA Soil Kit  following the manufacturer’s instructions. The quality of the extracted DNA was verified using 1.0% agarose gel electrophoresis and a NanoDrop ND-1000 spectrophotometer . All extracted DNA samples were stored at −20°C until analysis.The full-length sequences of 16S rRNA from genomic DNA were amplified using barcoded universal primers 27F and 1492R. Despite the wide use of ITS rRNA as a fungal DNA barcode, its ability to resolve relationships at higher taxonomic levels is inferior to that of many protein-coding genes. Therefore, we combined ITS with the large rRNA subunit (28S rRNA) to differentiate target organisms from close phylogenetic neighbors , 63. The18 SB-Aq chromatographic column , solution volume ratio  of 3:97, and UV detection wavelength of 210 nm [The organic acid compounds in koumiss were quantified using a high performance liquid chromatography (HPLC) system  with a Cf 210 nm .g at 4 °C for 10 min. The supernatants were analyzed using the SA402B taste-sensing system . A random sample placed in the first position was used as the control.The koumiss samples were diluted 1:1 with double-distilled water and centrifuged at 1700×p < 0.05. Spearman’s rank correlation was used to determine the relationships among species, flavor, and organic acids. Graphs were generated using the ‘ggplot2’ R package  and GraphPad Prism software .The data are presented as means ± standard error (SE). Statistical analyses were performed using R software . Statistical differences between two groups were assessed using the Wilcoxon rank sum test. Differences were judged to be statistically significant when Additional file 1: Figure S1. Radar curves for koumiss taste data. The red refer to Xilinhot-Urban samples, and blue refer to Xilingol-Rural samples.


Brassica juncea is a major oilseed crop in tropical and subtropical countries, especially in south-east Asia like India, China, Bangladesh, and Pakistan. The widespread cultivation of genetically similar varieties tends to attract fungal pathogens which cause heavy yield losses in the absence of resistant sources. The conventional disease management techniques are often expensive, have limited efficacy, and cause additional harm to the environment. A substantial approach is to identify and use of resistance sources within the Brassica hosts and other non-hosts to ensure sustainable oilseed crop production. In the present review, we discuss six major fungal pathogens of B. juncea: Sclerotinia stem rot (Sclerotinia sclerotiorum), Alternaria blight (Alternaria brassicae), White rust (Albugo candida), Downy mildew , Powdery mildew (Erysiphe cruciferarum), and Blackleg (Leptoshaeria maculans). From discussing studies on pathogen prevalence in B. juncea, the review then focuses on highlighting the resistance sources and quantitative trait loci/gene identified so far from Brassicaceae and non-filial sources against these fungal pathogens. The problems in the identification of resistance sources for B. juncea concerning genome complexity in host subpopulation and pathotypes were addressed. Emphasis has been laid on more elaborate and coordinated research to identify and deploy R genes, robust techniques, and research materials. Examples of fully characterized genes conferring resistance have been discussed that can be transformed into B. juncea using advanced genomics tools. Lastly, effective strategies for B. juncea improvement through introgression of novel R genes, development of pre-breeding resistant lines, characterization of pathotypes, and defense-related secondary metabolites have been provided suggesting the plan for the development of resistant B. juncea. Brassica juncea (L.) Czern & Coss is a natural amphidiploid  of Brassica rapa  and Brassica nigra  belonging to this family and cultivated worldwide for its edible oil. The estimated area, production, and yield of rapeseed-mustard in the world were 36.59 Mha, 72.37 Mt, and 1980 kg/ha, respectively, during 2018–2019. India contributed 19.8 and 9.8% to global acreage and production, respectively  Sacc.; A. brassicicola (Schwein.) Wiltshire; A. raphani Groves & Skolko], Sclerotinia stem rot [Sclerotinia sclerotiorum (Lib.) deBary], White rust [Albugo candida (Pers.) Kunze], Clubroot [Plasmodiophora brassicae Woronin], Powdery mildew [Erysiphe polygoni DC.], Blackleg [Leptosphaeria maculans (Desmaz.) Ces.& De Not.], and Downy mildew [Hyaloperonospora parasitica (Pers.: Fr.) Fr.]  Fr.] . These dr.) Fr.] . Fortunar.) Fr.] .The plants respond to pathogens either through constitutive or inducible defense mechanisms to protect themselves from damage. Constitutive defense includes preformed physical barriers such as cell walls, cuticle, wax on the outer surface, bark, and cork layers. Alternately, plants produce toxic chemicals, cell lytic enzymes, and deliberate cell suicide when attacked by pathogens, which form the inducible defense systems. The inducible defense system of the plants activated only after a pathogen is recognized by his immune responsive system because of the production of chemicals or proteins. This leads to high energy costs and nutrient requirements in association with their production and management. A lot of plant pathogens, known as biotrophs, make a close connection with their host plant suppressing its defense system but keeping their host alive, use the plant’s nutrients for their growth and reproduction. However, some pathogens produced toxins, cell degrading enzymes, or proteins to overcome plant defense systems promoting the rapid release of nutrients called necrotrophs. The pathogens can infect more than one host plant called the host range. Similarly, plants can defend themselves against a particular, or wide range of pathogens, or races of the pathogen. This type of plant defense is called vertical and horizontal resistance, respectively. The vertical resistance of the host plant can protect from a particular type of pathogen or race with high intensity but does not last long against the pathogen. The vertical resistance of the host plants is due to one or two major genes and is highly specific. However, the horizontal resistance is durable and it can protect host plants against a wide range of pathogens and races. In this, a group of genes simultaneously take part in host defense and present within the QTL. The genes that take part in the host defense systems are called R genes. The plant pathogenic fungi and bacteria have Avr (avirulence) genes to identify their host plants and infect them. The Avr genes produce small secreted protein (SSP) effectors and secondary metabolites (SM), the key elements of their pathogenesis that regularize innate immunity of the host plant and facilitate infection. However, the host resistance can be established only when the R gene of the host recognizes the effectors or secondary metabolites. This recognition usually triggers its defense responses including the hypersensitive response (HR) and results in resistance from the plant to the pathogen .cis-jasmone, Jasmonyl isoleucine (JA-Ile), and Jasmonoyl ACC (JA-ACC). The role of free JA and Me-JA is well established under various biotic and abiotic stress conditions , Salicylates (SAs), Brassinosteroids (BR), Ethylene (ET), Abscisic acid (ABA), and Phosphoglycerolipids (PGL). Out of all the above signaling pathways, JAs and SAs are reported most effective against pathogenic diseases. The polyunsaturated fatty acids (PUFA), a group of plant oxylipins produced Jasmonates (JAs) through oxidation by using one of the seven different branches of the lipoxygenase (LOX) pathway. However, the key component of JA biosynthesis is allene oxide synthase (AOS) that utilizes 13-hydroperoxide from α-linolenic acid . PUFA is released from the chloroplast membrane and converted into 9 or 13-hydroperoxides through the LOX pathway, which further enters into the AOS pathway to produce free JA. There are a lot of JA derivatives present in the host plants such as free JA, methyl-jasmonate (Me-JA), nditions . Salicyl tobacco . The key tobacco . The hor tobacco  exhibite tobacco . Very feB. juncea by focusing on major pathogens and corresponding diseases such as Sclerotinia stem rot, Alternaria blight, White rust, Downy mildew, and Powdery mildew  deBary is a necrotrophic fungal pathogen causing stem rot disease in B. juncea worldwide  identified a large number of single nucleotide polymorphisms (SNPs) linked to resistance in A03, A06, and B03 chromosomes. The annotation studies identified an array of resistance mechanisms, i.e., signal transduction pathways, hypersensitive responses, and production of anti-fungal proteins and metabolites. A total of five SNPs on the A03 chromosome were found to be associated with LRR-RLK genes that encoded LRR-protein kinase family proteins. They also predicted genetic factors associated with pathogen-associated molecular patterns (PAMPs) and effector-triggered immunity (ETI) on the A03 chromosome. These belonged to three R-Genes encoding TIR-NBS-LRR proteins. Hitherto, no resistant gene has been cloned for this disease  with the susceptible B. oleracea var. alboglabra was used to identify QTL for SSR. The F2 population exhibited a total of 12 and 6 QTLs for leaves and stem resistance, respectively. The candidate R genes were identified for Sclerotinia resistance on C09 of B. oleracea by blasting the sequence of adjacent markers to the B. rapa reference genome. The 12.8-cM genetic region on C09 contains two major QTLs for both leaf and stem resistance, while the corresponding 2.7-Mb genomic region on chromosome A09 of B. rapa harbors about 30 genes putatively encoding resistance-related and defense associated proteins. The putative genes present on the A09 chromosome belonged to the CC-NBS-LRR class  , C (moderately virulent), and D (avirulent) collected from all over India (A. brassicae were examined by Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) markers for genetic variability. The results of the study revealed that there was no clear grouping of isolates but little variations were recorded which may be due to ecological or survival-based adaptations  . The bliin India  and 32–5in India . The patin India , India  carry the inoculum from one crop season to another  during infection characterized as ABR toxin protein  and 6-methoxycamalexin (C H N SO)  in Capsella bursa-pastoris  sinalbins A and B in the leaf and stem tissues for resistance against the A. brassicae ( surface . The C.  H N SO) . The patpastoris . The mutsistance . Howeverpathways , 2001. T. juncea . The S. rassicae . The varrassicae .B. juncea and S. alba. The transcripts of all five defense-related genes accumulated locally as well as systemically at a greater level and earlier in S. alba than in B. juncea upon challenge inoculation with A. brassicae. The PDF1.2 was induced by SA and PR-1 by JA in both B. juncea and S. alba resulting in both JA and SA responsive genes conferring resistance against A. brassicae in S. alba. Similarly, upon challenging with A. brassicicola, the S. alba plants showed higher accumulation of abscisic acid and JA than B. juncea  was developed and evaluated against A. brassicae infection under glasshouse conditions. It was found that transgenics showed higher resistance levels for diseases such as longer incubation and latent period, smaller necrotic lesion size, lower disease intensity, and delayed senescence  and type I ribosome-inactivating protein  to coexpress. These transgenics were evaluated in vitro and greenhouse conditions for resistance against A. brassicae. The study revealed a 44% reduction in hyphal growth, delayed onset of the disease, and a limited number of lesions as compared to wild resistant plants , MYC3 (JAZ-interacting transcription factor), ERFs (Ethylene response factors), BRG (Botrytis-induced Related Gene), RBOHD (Respiratory Burst Oxidative Homolog D), NPR3 (Non-expressor of PR3) including 14 defensin and defensin-like genes known for antimicrobial/antifungal activities. The exogenous application of β-Aminobutyric Acid to B. juncea prevented plants from A. brassicae infection independent of SA and JA signaling pathways  Kuntze. is an obligate parasite of the oilseed Brassicas causing white rust and staghead disease worldwide including Brazil  is produced in A. candida infected plants which lead to hyperplasia and hypertrophy of leaf, stem, and floral parts  markers At5g41560 and At2g36360 derived from A. thaliana were successfully validated to have a close link with white rust resistant loci AcB1-A4.1 and AcB1-A5.1 of B. juncea, respectively  for resistance against A. candida. The RAC genes belong to the Drosophila toll/interleukin-1 receptor (TIR) nucleotide-binding site leucine-rich repeat (NB-LRR) class of plant resistant gene  . The expression of WRR4 in susceptible lines of B. juncea and B. napus provided complete resistance against white rust pathogen belonging to the races 2 and 7, respectively (ectively . The mapant gene . Anotheron: Col)  which enectively .B. juncea  can be explained in A. thaliana accessions by at least one of four genes  encoding nucleotide-binding, leucine-rich repeat (NLR) immune receptors. However, the WRR12 gene identified in A. thaliana confers resistance to A. candida race 9 that infects B. oleracea. Thus, the effector-triggered immunity conferred by the distinct NLR-encoding genes in multiple A. thaliana accessions provides species-wide resistance to different races of A. candida (B. juncea—Donskaja-IV from the east European gene pool line was mapped for conferring resistance locus (AcB1-A5.1) against white rust and was found to be completely resistant to six different Indian isolates. The locus was identified to possess a single CC-NB-LRR protein-coding R gene (BjuWRR1) which provided resistance to genetically transformed susceptible Indian mustard variety Varuna from all the isolates (B. juncea var. Tumida (resistant) × B. juncea var. Varuna (susceptible) was identified with a new white rust resistance-conferring locus on LG A6 which was most likely to BjuA046215 candidate gene, a CC-NBS-LRR type R gene, and closely related to BjuWRR1  Constant. Syn. H. brassicae which causes heavy yield loss to cruciferous crops and is distributed worldwide. In India, the disease is responsible for upto 66% yield loss in B. juncea making up a yearly loss of about 683.1 million INR, depending on the disease severity  highly resistant to downy mildew disease in all 3 years. The F2 population developed from the resistant and susceptible genotypes of broccoli  and cauliflower (B. oleracea L. var. botrytis) showed segregation in a 3:1 (resistant:susceptible) ratio which indicated that the resistance is governed by a single dominant gene and also confirmed by test-cross  of nt genes . In simiant gene . Dang etst-cross . It was ant gene , 2004.B. oleracea  identical to BraDM were mapped to A08 through three SNP markers  for resistance at the seedling, young plant, rosette, and heading stages . They concluded that resistance for powdery mildew is not simply due to constitutive activation of the salicylic acid or ethylene and jasmonic acid-dependent defense pathways, but because the mutants did not constitutively accumulate elevated levels of PR1 or PDF1.2 mRNA.The powdery mildew of Brassicaceae is caused by biotrophic fungal pathogen h Africa  in dry-warvensis . The patarvensis . Tonguc B. napus conferring broad-spectrum resistance for powdery mildew pathogen. One and three genes homologous to RPW8 were present in B. rapa and B. oleracea, respectively. There should be seven homologs of RPW8 in B. napus. They found that the copy of B. oleracea resistant genes was highly conserved, while the B. rapa homolog was variable in the B. napus genome possibly due to gene loss, point mutation, insertion, deletion, and intragenic recombination. B. juncea accessions against E. cruciferarum under natural hot spot conditions. They have identified only one accession (RDV29) consistently resistant for 5 years. This line was used for hybridization with a highly susceptible line (RSEJ775) to obtain filial and backcross populations. The study revealed that resistance was governed by two different semi-dominant genes. A. thaliana accessions to identify resistant loci for powdery mildew pathogen (E. cichoracearum). Their study revealed that out of six, five accessions conferred resistance through a single locus and all were independent and the another one accession have resistance from two unlinked loci. A. thaliana for resistance to powder mildew. The majority of R genes characterized in A. thaliana for powdery mildew disease belong to the nucleotide-binding site and C-terminal leucine-rich-repeats (NB-LRRs) and a less prevalent N-terminal transmembrane domain and a coiled-coil motif superfamily. These genes were identified as NDR1, EDS1, PAD4, NPR1, EDS5, RAR1, SGT1b, RPW8, PBS3, COI1, EIN2, and EDR1  or its cell-free culture filtrate in B. napus and R. alboglabra to powdery mildew pathogen. The results of the study revealed that the pathogen failed to develop colonies on R. alboglabra leaves even after 10 days of inoculation while B. napus leaves have fungal colonies after 6 days of inoculation. The expression of PR-1 and PR-2 levels increased in E. cruciferarum infected leaves but decreased in the TH12 treated leaves. However, the expression of PR-3 and PDF1.2 is decreased in E. cruciferarum infected plants whereas it was increased when treated with TH12 suggesting that TH12 can be used for improving resistance to powdery mildew in hosts.and EDR1 . However disease . AlkooraLeptosphaeria maculans (Desm.) Ces et de Not. [anamorph: Phoma lingam (Tode: Fr.) Desm.] and L. biglobosa sp. nov., Shoemaker & Brun which belongs order Ascomycota  of the pathogen are formed as small dark spots on leaves and stem which contains pycnidiospores that transmitted through water splashes  for the presence of resistance to blackleg disease. They have identified a total of four co-segregating RFLP markers for cotyledon and adult-leaf resistance which was associated with six loci present on linkage groups 2, 5, and 8. A triplicate region in the B- genome had preserved the resistant loci in all three species. B. napus suffer more severely from the blackleg disease than B. juncea (B. napus from members of the Brassicaceae family (B. nigra monosomic lines for chromosome 4 in B. napus have the same level of resistance as in B. nigra (2 and backcross population of two different cultivars of B. juncea (resistant- AC Vulcan and susceptible- UM3132) was segregated for blackleg resistance which suggested that the resistance was controlled by two independent dominant and recessive genes positioned on linkage group J13 and J18, respectively . The gene was localized to a 5.8 cM interval corresponding to approximately 873 kb of the B. napus chromosome A10  for blackleg in B. napus through NGS . Mapping population contributed about 35 percent phenotypic variations.The ough NGS . The LmR of LmR1 . Yu et aass 400” . Raman esistance . Huang eB. rapa and B. nigra) of B. juncea have evolved from a common ancestor followed by ancient genome triplication along with structural and numerical changes about 7.9–14.6 million years ago  while B. napus formed about 0.038–0.051 mya, slightly after the formation of B. juncea. B. juncea was estimated by flow cytometry to possess a 922 Mb genome size. The B. juncea genome has 316.1 Mb of repetitive sequence, out of which 131.2 Mb are from B. rapa and 216.5 Mb from B. nigra genomes. During the speciation process, it was estimated that a total of 562 and 545 genes from B. rapa and B. nigra subgenomes of B. juncea were lost, respectively, as compared to their common ancestral genomes. This number is higher than the gene loss estimated in B. napus subgenomes (BnaA and BnaC) as compared to their common ancestral genomes. However, the number of genes lost in B. juncea and B. napus was consistent since their formation. The A subgenome of B. juncea and B. napus had divergent origins. It was discovered that A subgenome of B. juncea might be derived from Asian B. rapa ssp. tricolaris, while the subgenome A of B. napus might be derived from European B. rapa ssp. rapa. Thus, both A subgenomes of allotetraploids had independent geographical origins. It was also discovered that the chromosomal regions of B. juncea had gone through various rearrangements and formed the current species contributing to gene duplications and losses. The polyploidy nature of B. juncea played an essential role in genome speciation and plasticity  and the Bc subgenome from the African oil crop B. carinata (BcBcCcCc) and synthesized allohexaploid (ArArBcBcCcCc), crossed with traditional B. juncea to generate pentaploid F1 hybrids (ArAjBcBjCc), with subsequent self-pollination to obtain newly synthesized B. juncea (Ar/jAr/jBc/jBc/j). The genetically stable new type of B. juncea population was obtained at the F6 generation retaining good fertility and rich genetic diversity while being distinct from the traditional B. juncea. This newly developed B. juncea had more than half a modified genome due to exotic introgressions and novel variations in gene copies, numbers, and sequences . A total of 4 and 7 QTLs for the white rust and blackleg resistance in B. juncea respectively, were identified. The white rust resistant locus AcB1-A04.1 was present on the linkage group A04 and B01, while another locus AcB1-A05.1 was present on the same linkage group on an overlapping position at A05 with a similar copy at B06. Similarly, blackleg-resistant quantitative loci-PhR2 were identified on the linkage groups A03 and B03, and another QTL, RLM6, identified on three linkage groups . However, PhR2 and LMJR1 resistant loci for blackleg were present on linkage group B03. This study concluded that the B. juncea genome has duplicated sequences , a total of seven pathogenic races were identified designated as Abr1 to Abr7  and analyzed them with RAPD and RFLP markers. Having tested a total of 20 decamer primers of arbitrary nucleotide sequences for PCR amplification of A. brassicae genomic DNA, only five primers with amplified genomic DNA from 20 isolates of A. brassicae were selected and classified into four groups. However, the variations between isolates collected from intra-geographical regions were very less apparent. A. brassicae collected from a wide geographic area of north-west India. Out of these, 114 were identified as pathogenic and all were categorized under seven broad groups based on morphological characters showing a wide range of diversity. A. brassicae collected from different parts of India through 100 RAPD decamer primers and found genetic variability among the isolates at the genomic level, but not in the highly conserved regions of the genome of A. brassicae. A. brassicae collected from eight north Indian states and found a total of 57–78% genetic diversity within all isolates with no correlation established. They determined the genetic relationship among 32 A. brassicae isolates by UPGMA revealing the clustering into four major classes that were further subdivided into nine subgroups. A. brassicae specific genes which included many secreted proteins and effectors. They also identified gene clusters responsible for the production of pathogenicity specific Destruxin-B (a cyclic depsipeptide). However, in the recent past, a chromosome level about a complete genome assembly of A. brassicae was developed that could be useful to uncover the B. juncea–A. brassicae pathosystem related genes (The genome complexity of of crops . However to Abr7 . The raced genes .A. candida) isolated from four Brassicaceae members behaved as four independent races in Saskatchewan (Canada). These races were evaluated for their virulence in various Brassica members collected from different parts of the world. The results showed that certain crop species were susceptible to specific race but not to all races. However, some accessions of Brassica show resistance to some of these races, but not all  RH, >97 mornings (maximum) RH with 16–24°C daily temperatures. However, the staghead formation was influenced positively between temperature ranges of 20–29°C and >97% morning RH (B. juncea needs warmer (24–30°C) environmental conditions and low relative humidity (∼65 RH) for successful disease establishment and conidial dispersion , and inoculums dispersal conditions. All these environmental factors directly influence the host-resistance status by affecting pathogen growth and reproduction potential. The sclerotia developed by severely . The carseverely . The devseverely . The spoeratures . For sucm 4 days . The sevrning RH . In contspersion .A. candida was identified on Lepidium latifolium (pepperweed) in California which was unable to infect fifteen host differentials of the Brassicaceae family. Thus, the host range information of any particular pathogen is important to identify new races  and B. spinescens through ovary, ovule, and embryo rescue conferring resistance for Alternaria blight. Moreover, the somatic hybridization (protoplast fusion) technique has been used for transferring resistance from S. alba to B. napus  seedlings with avirulent isolates of P. parasitica induced systemic resistance against virulent isolates and reduced disease incidence upto 70% , however, B. juncea still possesses resistance to mildew pathogen. The pre-infection with A. candida can successfully suppress a broadspectrum resistance conferred by RPW8 to the two morphotypes of Erysiphe spp. (a powdery mildew pathogen) in A. thaliana  that are present in the cell membranes have detergent-like properties and disintegrate cell membranes of fungal pathogens. However, some fungal pathogens have saponin-degrading capacity causing diseases like Botrytis cinerea, Fusarium oxysporum, and Septoria lycopersici. The phytoalexins  have antibacterial and antifungal properties that are produced on the pathogenic attack. These are toxic molecules that disturb the metabolism or cellular structure of pathogens but are often toxic to a specific pathogen  and defense-related proteins  to protect them from pathogens or stressful conditions other than occupying resistant genes . The propathogen . The glull lysis . The reall lysis .Hordeum vulgare and Triticum aestivum. They are predominantly characterized in the seeds but can be present in all types of plant tissues . The specific mechanisms employed by defensins to inhibit fungal growth are still being characterized, but they appear to act upon molecular targets in the plasma membrane of pathogens. The chitinases and glucanases are enzymes that catalyze the degradation of chitin and glucans, respectively, polymers with a backbone similar to cellulose that is present in the cell walls of true fungi. The in vitro analysis has confirmed the anti-fungal activities of these enzymes, and transgenic plants expressing a high concentration of these enzymes exhibit increased resistance to a wide range of pathogens . Moreover, the transgenic A. thaliana plants expressing ARACIN1 were well protected from infections of B. cinerea and A. brassicicola (The defensins are small cysteine-rich proteins that show broad-spectrum anti-microbial activity and were first isolated from the endosperm of athogens . Anotherathogens . The recsistance . Thioninsistance . These asistance . The trasistance . A largeRaphanus . The KelB. napus . It was ca crops . The in sicicola .A. alternata can be identified by the comparative analysis of ITS, GAPDH, and RPB2 genetic markers (A. brassicae which were absent in all other Alternaria species. These genes have included 35 secreted protein-coding genes and out which 11 were predicted to be effectors. A large number of these proteins were uncharacterized proteins with no known function. These A. brassicae specific genes and effectors could be a potential source for categorization and identification of A. brassicae races.The identification of pathogen race or strain is important for the successful management of disease outbreaks in a particular region/country as some resistant genes were identified to work against a particular race or strain. This goal can be achieved by the characterization of pathotypes through genetic markers such as race/strain/isolate specific molecular markers, proteins, and genes. The strains of  markers . RajaramThe advanced sequencing technology provides near-complete genome assemblies that serve better in gene predictions and will also provide improved results for structures and functions of effector proteins. The characterization of effector genes/proteins can be a potential identification source for pathotypes . HoweverSclerotinia sclerotiorum carried SCFE1 elicitor which evokes MTI responses and is sensed by RLP30, which confirms the association of MTI in resistance to S. sclerotiorum (S. sclerotiorum. On being challenged by Sclerotinia, these elicitors can activate the plant immune system against the invading pathogen (b-aminobutyric acid, 2, 6-dichloro-isonicotinic acid (rotiorum . Some otrotiorum , SsCut (rotiorum , and SsSrotiorum , have alpathogen . A largepathogen , Chitosanic acid , methyl nic acid , arachidnic acid  were appB. juncea against major fungal pathogens is a difficult procedure owing to the close association of R genes with other agronomic traits, variations in host resistance-response against pathogenic races, the polygenic inheritance of resistance, and gene interactions. Also, the identification of R genes is cumbersome because of factors like genetic variations in host subpopulations and pathotypes, the emergence of new pathotypes, genomic tools used, environmental factors, resistance evaluation techniques, scoring criteria, etc. However, continuous efforts are being made to identify R genes or quantitative trait loci for major fungal diseases of Brassica crops. A stable and long-lasting resistance source is a prerequisite for resistance breeding programs. The cloning of previously characterized R genes in Brassica will help in the identification of plant defense mechanisms that support the search of novel R gene sources. The studies on host–pathogen interactions are important to have an insight into the pathogenesis and response of the host against pathogen infection. The genome assembly database played a vital role in identifying novel R genes as the advanced sequencing technologies provide near-complete genome assemblies of diploids or polyploids. This may allow us to identify race-specific or non-specific R genes.The identification and introgression of resistant gene sources into B. juncea for improvement through advanced molecular techniques.There is a need for universally accepted robust screening and scoring methods for each host-pathogen systems so that true R gene sources could be identified. To increase host resistance for wider pathogen races, gene pyramiding provides durable resistance and sustainable establishment of crop plants in disease-prone areas. Improving knowledge about elicitors and effectors is also important to develop successful disease management strategies. The development of novel pre-breeding material from close or distant resistant wild relatives and marker-assisted selection approach may help to efficiently facilitate Brassica-improvement programs. This review presents a comprehensive analysis of the R gene sources and their utilization in KS and PK conceptualized and drafted the manuscript, prepared the tables and figures. PK and PR edited and finalized the manuscript. All authors read and approved the final manuscript.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.


Trichophyton mentagrophytes and Microsporum canis were identified by using mycological examinations. To our knowledge, this is the first case of kerion caused by the combined Trichophyton mentagrophytes and Microsporum canis. Treatment with oral terbinafine for 2 months was effective.A 5-year-old boy was presented with large ulcer accompanied by surrounding follicular pustules on the left parietal scalp. Dermoscopy showed “comma” and dystrophic broken hairs. Fungal culture showed mixed growth of two types of colonies.  The distribution of causative species of dermatophytes varies greatly in different geographic regions. For example, nd China ,2, while America . Kerion  America . And adu America . A singlnt years ,5. It ha2A 5-year-old boy presented with a large ulcer accompanied by surrounding follicular pustules on the left parietal scalp (day 0). Two weeks prior, the lesions appeared initially as pruritic follicular pustules, which increased gradually in size and formed an abscess. Before appearance of these lesions, he had experienced scalp scratch. He initially underwent incision and drainage of the abscess in other hospital and was treated with oral amoxicillin. A large ulcer appeared subsequently at the primary pustule site, and he presented to our hospital for further treatment. Besides, he lives in the rural area and keeps pets including cat and rabbit. Physical examination revealed a 3 × 4 cm ulcer with several peripheral follicular pustules on the left parietal scalp A. Left chttp://www.wi.knaw.nl/Collections/BioloMICSSequences.aspx?expandparm=f&file=FUNGI&file=FUNCBS&file=YEASTseq&file=MOLDseq&file=UNITE&file=Yeast&file=Fusarium&file=Dermato&file=indoor&file=Morch&file=ITSSeq&file=Bipolaris&file=CGattii&file=CNeof&file=PJ&file=Scedo&file=FunBOLD&file=PYCC&file=EFSeq&file=Mycokey&file=PasteurMOULD&file=PasteurYEAST). Molecular identification for these 2 isolates suggested identity to T. mentagrophytes and M. canis. Taking these data together, the two isolates were identified as T. mentagrophytes and M. canis, respectively. Therefore, a diagnosis of kerion caused by a combination of T. mentagrophytes and M. canis was finally established.Dermoscopic examination showed large patches of erythema, scales, “comma” hairs, and dystrophic broken hairs with white hair casts near the scalp by using a hand-held dermoscope connected with digital camera (day +1) . Direct The patient was treated with oral terbinafine (125 mg per day) combined with topical 2% ketoconazole cream for two months. In parallel, oral prednisone (20 mg per day) was administered for two weeks to suppress inflammation. The lesions improved significantly, leaving residual scarring alopecia B, and no3T. tonsurans or M. canis [TC is a common scalp disease in the worldwide, most often caused by M. canis . Kerion M. canis . Kerion M. canis . TherefoDermoscopic examination is a rapid, non-invasive and painless method for the diagnosis of TC . In addiT. mentagrophytes and M. canis was diagnosed.This patient was initially misdiagnosed as bacterial infection and underwent incision and drainage of abscess that made TC aggravated. After mycological examinations in our hospital, kerion caused by combined Microsporum infections [Trichophyton spp. than Microsporum spp [Microsporum and Trichophyton species, and is commonly used as second-line therapy [The aim of the treatment is to eradicate the organism to achieve both clinical and mycological cure as quickly and safely as possible . Treatmefections ,15. Howe therapy . In addi therapy . TherefoMycobacterium leprae and Cryptococcus [Aspergillus fumigatus and Candida albicans [T. mentagrophytes and M. canis. Therefore, we conclude that clinicians should be aware of TC that can be rarely caused by mixed dermatophyte infection, and that dermoscopic and mycological examinations are clinically useful in differentiating between kerion and other scalp diseases.Recently, the occurrence of mixed infection is a growing public health problem, including bacteria and fungi such as tococcus , differealbicans . TC is ralbicans ,20. Our There are none.


Komagataella phaffii.Eukaryotes use distinct networks of biogenesis factors to synthesize, fold, monitor, traffic, and secrete proteins. During heterologous expression, saturation of any of these networks may bottleneck titer and yield. To understand the flux through various routes into the early secretory pathway, we quantified the global and membrane-associated translatomes of PMA1 sequester more ribosomes at the ER membrane than any others. Comparison to Saccharomyces cerevisiae reveals conservation in the resources allocated by gene ontology, but variation in the diversity of gene products entering the secretory pathway.By coupling Ribo-seq with long-read mRNA sequencing, we generated a new annotation of protein-encoding genes. By using Ribo-seq with subcellular fractionation, we quantified demands on co- and posttranslational translocation pathways. During exponential growth in rich media, protein components of the cell-wall represent the greatest number of nascent chains entering the ER. Transcripts encoding the transmembrane protein A subset of host proteins, particularly cell-wall components, impose the greatest biosynthetic demands in the early secretory pathway. These proteins are potential targets in strain engineering aimed at alleviating bottlenecks during heterologous protein production. Komagataella phaffii  standpastoris , 11. Thuecretion . Indeed,analysis  and implanalysis , 14. Sucanalysis , 16.K. phaffii, an early bottleneck is the translocation of newly made proteins from the cytoplasm into the lumen of the endoplasmic reticulum (ER) [sec translocon. At least three major translocons exist in yeasts , which can accept proteins as they are synthesized by ribosomes  or after synthesis of the polypeptide chain is complete . Besides translocon architecture, co- and posttranslational pathways differ in their reliance on cytosolic molecular chaperones [sec translocon is a major point of entry for integral membrane proteins of the endomembrane system [sec translocon require SRP for targeting to the ER over mitochondria [Identifying and relieving protein biogenesis bottlenecks is one strategy to improve yields of high-value, recombinant proteins , 17. Forlum (ER) , 19. Yealum (ER) ). In theaperones , 21. Traaperones . Some siaperones ; these taperones , 25. Bine system . Integrachondria .sec translocons represent limited pools of resources that are distributed between heterologous proteins and the host proteome [K. phaffii has been characterized under several conditions [S. cerevisiae. [K. phaffii.For any production host, ribosomes, molecular chaperones, and proteome –29. Unliproteome , the comnditions , but thenditions . Per molnditions , 34. Howevisiae. , 35, 36 K. phaffii? Which host genes produce the most nascent chains, competing for chaperones and sorting factors within the endomembrane system? To answer these questions, we quantified active translation globally and at the surface of the ER or mitochondria. Our analysis reveals the set of proteins that enter the secretory pathway cotranslationally and predicts the set that enter posttranslationally. In each set, we estimate demand for ribosomes and translocons. We distinguish between resources that act on a per nascent chain basis from machinery that is utilized based on elongation time.Which host proteins sequester the most biogenesis machinery in the early secretory pathway of Komagataella phaffii GS115 (Invitrogen). For each Ribo-seq biological replicate, 500 ml liquid cultures of YPD  were grown to an S. cerevisiae fractionated Ribo-seq data [All experiments were performed using seq data , cyclohenscripts . Short inscripts .g for 10 min. Supernatants from samples lysed with membrane lysis buffer were collected and used as “total” fractions. Supernatants from samples lysed with soluble lysis buffer were collected and used as “soluble” fractions. The pellets from sample lysed with soluble lysis buffer were resuspended in Cells were lysed in either soluble lysis buffer (Lysed samples were digested using 40 U of ribonuclease A (Ambion) for Purified fragments were used to prepare sequencing libraries as described in  with somCells were grown in YPD at 30 °C with agitation to an A novel transcriptome was assembled using data derived from Ribo-Seq, long-read RNA-Seq, and a prior genome sequence of strain GS115 . A flowcimmerHMM  and CodiimmerHMM , which wimmerHMM  was used package .Komagataella pastoris GS115 [Ribo-seq reads were mapped to the genome of is GS115  using HIis GS115 , 50. Aliis GS115 . Mapped is GS115  and convis GS115 . Each reis GS115 .Read counts were normalized at the codon level using a metagene analysis that provides a global profile for each data set. First, for each ORF, reads at each codon position were scaled by the average reads per codon mapped ORF. Then, for codon position, either a mean or median value was calculated from all ORFs using the following scheme: for positions 1 to 100, a rolling mean with a window of 10 codons; for positions 100 to 1000, a rolling mean with a window of 100; for positions 1000 and onward, a rolling median with a window of 1000. In calculating corrected transcripts per million (cTPM), codon read counts were scaled by dividing the metagene-derived value at that position and normalized by their pseudo gene lengths  and a per million scaling factor unique to each data set. In calculating ribosomes per million (cRPM), a ribosome scaling factor was created for each gene by dividing the sum of the metagene-derived values at all codon positions by the sum of smoothed reads per codon with the mask applied . The ribosome scaling factor is multiplied by unmasked gene read counts and normalized by a per million scaling factor unique to each data set to give RPM. Membrane enrichment is quantified for each gene as the K. phaffii transcripts through homology. Firstly, transcripts were assigned names inherited from S. cerevisiae using BlastP [S. cerevisiae or known ascomycetes were assigned names inherited from K. phaffii GS115 [Gene names were hierarchically assigned to novel g BlastP  with an g BlastP  using a ii GS115  using BlORFs were classified by function, cellular location, and sequence features using various prediction software. Functions were assigned ontologically using clusters of orthologous groups (COG) and were prepared using EggNOG 4.5 . VironoiK. phaffi using the S288C reference genome R64-2-1 [Ribo-seq data for total protein synthesis were taken from , and dat R64-2-1 .K. phaffii GS115. We asked which genes were responsible for sequestering limited biosynthetic resources, such as ribosomes and ER translocons. We also asked which genes were responsible for producing the most nascent chains, which is critical for predicting amino acid usage, as well as modifications that act on a per chain basis . Ribo-seq provides a snapshot of protein translation, allowing us to answer both of these questions [We sought to globally quantify several aspects of protein synthesis in uestions . It is auestions , which aWe noticed that ribosome-protected read patterns were often inconsistent with prior annotations of open reading frames , even on transcripts encoded on the same DNA strand. As a result, methods that construct transcripts from short-read sequencing merge data from adjacent genes into a single transcript. We therefore collected long-read data using Oxford Nanopore PCR-cDNA sequencing and developed a pipeline to integrate Ribo-seq, long-read RNA-seq, and de novo gene prediction S. cereviproteins , 36  and PGK1 [S. cerevisiae generates a similar amount of nascent chains to the same function, but it does so using a combination of its paralogous genes TEF1 and TEF2. Unsurprisingly, Crabtree-positive S. cerevisiae generates three times more polypeptides involved in glycolysis and fermentation than K. phaffii .Of the 5329 paralogs . The inf. phaffi , and ourand PGK1 . S. cereK. phaffii. Recovery of membrane associated mRNA strongly depends on active translation [Indeed, these two species also show divergence in energy production with regards to cotranslational mitochondrial import Fig. . Our subnslation . TherefoPDI1, encoding an ER lumenal protein-disulfide isomerase that is essential for ER homeostasis. Mitochondrially localized proteins have greater membrane enrichment in K. phaffii, which may be related to the greater use of aerobic respiration compared to S. cerevisiae .We next asked whether ER translocation pathways are conserved between the two species. Between homologs, membrane enrichment scores correlated with a Pearson’s r of 0.85 , ribosome demand (cRPM) and translocation pathway (membrane enrichment score) for ER destined proteins within the two species Fig. . In S. cS. cerevisiae [The yields of engineered, recombinant proteins are restricted by bottlenecks in biogenesis . Certainrevisiae . Other brevisiae . Translorevisiae , 85. Engrevisiae  and redurevisiae , providerevisiae , 88. OurS. cerevisiae that were collected under identical conditions [his4). Even under rich media with abundant extracellular histidine, this auxotrophy may influence gene expression compared to strains which supply HIS4. Future work involves quantifying demands at industrial scale in stirred bioreactors under induction of a heterologous protein. Second, we assume that elongation rates are relatively constant across genes. However, if the elongation rate is altered for a transcript, it may result in greater or fewer ribosome protected reads. We argue that on the whole, our assumption is valid, given that Ribo-seq accurately predicts mature protein stoichiometry [K. phaffii. However, our goal was to quantify the costs of protein synthesis, and so we argue that Ribo-seq is a more appropriate tool than mass spectrometry. Despite these limitations, our approach allowed us to interrogate protein translocation into the ER.Despite the ability of Ribo-seq to accurately quantify gene expression, our study has several caveats that limit interpretation. First, we have only considered yeast undergoing log phase growth in liter scale, aerated shaking cultures using rich media. This design enabled comparison to several published data sets using nditions , 62. We hiometry . Third, hiometry . Our ressec translocon [Most secreted proteins, including high-value targets like antibodies, will enter the ER via a anslocon . The traanslocon , 25, 36.anslocon  or the Eanslocon . Howeveranslocon , 92, theanslocon , 94. Furanslocon , 93. BecK. phaffii in this category , but also access to specialized cellular machinery. Our analysis in Additional file 1: Figure S1. Ribo-Seq models active translation. a. Distribution of reads for different length RNA fragments. b. P-site offset for 30 nt fragment reveals active translation.Additional file 2: Figure S2. Ribo-seq and long-read RNA-seq improve transcriptome annotation. Images are screen captures from Integrated Genome Viewer (MIT). a. Ribo-seq reads are stranded. In the top register, ribosome-protected footprint reads mapped to transcripts translated left to right are in red, and reads mapped transcripts translighted right to left are in blue. The middle register shows a prior annotation of transcripts and ORFs. The arrows indicate genes where the annotated translational start site disagrees with Ribo-seq. In both cases, an alternate start codon is used. The bottom register shows the annotation developed here using RNA-seq and long-read RNA-seq data. b. In an example transcript, Ribo-seq (top register) and long-read RNA-seq (bottomregister) reveal both the open reading frame and the untranslated regions (UTRs). c. Flow-chart of the annotation pipeline.Additional file 3 Annotation of K. phaffii GS115 transcriptome. GFF3 annotation file containing transcript structures derived from Ribo-seq and long-read RNA-seq analysis.Additional file 4. Comparison with prior K. phaffii annotations. Comma seperated value (CSV) file that links open reading frames defined in the current study with prior annotations of the K. phaffii transcriptome. Includes predicted protein sequence from each annotation. Additional details are provided in the file header.Additional file 5. Combined results of Ribo-seq analysis of K. phaffii. Read counts, corrected transcripts per million (cTPM) and corrected ribosomes per million (cRPM) scores for K. phaffii GS115 open reading frame (ORFs). The file also includes experimental membrane enrichment scores, predictions of ORF features , ORF sequences, homology to S. cerevisiae, and associations with prior annotations of the K. phaffii transcriptome. Additional details are provided in the file header.Additional file 6: Figure S3. Comparison of membrane enrichment between data sets. a. Comparing membrane enrichment in two Ribo-Seq data sets in K. phaffii. b. Comparing membrane enrichment in Ribo-Seq data sets in K. phaffii and S. cerevisiae.Additional file 7: Table S1. Biosynthetic demands for proteins with unknown functions by predicted subcellular localization.Additinal file 8. Combined results of Ribo-seq analysis of S. cerevisiae. Read counts, corrected transcripts per million (cTPM) and corrected ribosomes per million (cRPM) scores for S. cerevisiae open reading frame (ORFs). The file also includes experimental membrane enrichment scores, predictions of ORF features , and ORF sequences. Additional details are provided in the file header.Additional file 9: Figure S4. Comparison of metabolic burden for K. phaffii and S. cerevisiae. a. Total nascent chains for K. phaffii. b. Total nascent chains for S. cerevisiae.


Bulleyia Schltr. is monotypic and represented only by Bulleyia yunnanensis Schltr., native to the Yunnan of China, Bhutan, and northeast India. Here, we report the complete chloroplast (cp) genome sequence and the cp genome features of B. yunnanensis. The cp genome sequence of B. yunnanensis was 159,581 bp in length and presented a typical quadripartite structure consisting of one large single-copy region , one small single-copy region , and two inverted repeat regions . Besides, the cp genome encoded 132 genes, including 113 unique genes . The phylogenetic analysis suggested that B. yunnanensis be closely related to Pholidota in tribe Arethuseae. Bulleyia yunnanensis is a perennial epiphytic orchid with high medicinal value (Lu and Zhang Bulleyia was placed in tribe Arethuseae Lindl. of subfamily Epidendroideae (Orchidaceae), in which tribal relationships were unclear . The specimen was deposited in the Herbarium of Southwest Forestry University . The total genomic DNA was extracted from the fresh leaf using the modified CTAB procedure of Doyle and Doyle (Pleione formosana Hayata (GenBank accession number NC_042197) as the reference sequence, we assembled the cp genome from the clean reads by the GetOrganelle pipe-line  regions, a large single-copy region , a small single-copy region . This genome sequence encoded 132 genes, including 113 unique genes . Among them, the GC content of LSC, SSC and IR regions reached 35.2%, 30.4% and 43.3%, respectively. Besides, GC-content was 37.4% of the overall.The complete chloroplast genome of B. yunnanensis, a maximum-likelihood (ML) tree was constructed based on 78 protein-coding genes of nine species from four related tribes in subfamily Epidendroideae as ingroup, with two species from subfamily Orchidoideae as outgroup. These four tribes were selected according to the updated classification of Orchidaceae (Chase et al. http://www.phylo.org/, Miller et al. B. yunnanensis was closely related to Pholidota in tribe Arethuseae of Epidendroideae (To confirm the phylogenetic position of droideae .