Capsicum annuum and Capsicum baccatum was used to characterize population structure and species domestication of these two important incompatible cultivated pepper species. Estimated mean nucleotide diversity (π) and Tajima's D across various chromosomes revealed biased distribution toward negative values on all chromosomes (except for chromosome 4) in cultivated C. baccatum, indicating a population bottleneck during domestication of C. baccatum. In contrast, C. annuum chromosomes showed positive π and Tajima's D on all chromosomes except chromosome 8, which may be because of domestication at multiple sites contributing to wider genetic diversity. For C. baccatum, 13,129 SNPs were available, with minor allele frequency (MAF) ≥0.05; PCA of the SNPs revealed 283 C. baccatum accessions grouped into 3 distinct clusters, for strong population structure. The fixation index (FST) between domesticated C. annuum and C. baccatum was 0.78, which indicates genome-wide divergence. We conducted extensive linkage disequilibrium (LD) analysis of C. baccatum var. pendulum cultivars on all adjacent SNP pairs within a chromosome to identify regions of high and low LD interspersed with a genome-wide average LD block size of 99.1 kb. We characterized 1742 haplotypes containing 4420 SNPs (range 9–2 SNPs per haplotype). Genome-wide association study (GWAS) of peduncle length, a trait that differentiates wild and domesticated C. baccatum types, revealed 36 significantly associated genome-wide SNPs. Population structure, identity by state (IBS) and LD patterns across the genome will be of potential use for future GWAS of economically important traits in C. baccatum peppers.Principal component analysis (PCA) with 36,621 polymorphic genome-anchored single nucleotide polymorphisms (SNPs) identified collectively for  Capsicum spp.) are represented by at least 32 species, of which Capsicum annuum, Capsicum baccatum L. var. pendulum (Willd.) Eshbaugh, Capsicum chinense Jacq., Capsicum frutescens L., and Capsicum pubescens Ruiz & Pavon represent domesticated taxa  of peduncle length, an important domestication trait.Genotyping by sequencing (GBS) is a reduced representation method, which utilizes next-generation sequencing to develop genome-wide single nucleotide polymorphisms (SNPs). SNPs generated by GBS have been successfully deployed for genetic diversity analysis and Genome-wide association studies (GWAS) in several crops  collected from 32 countries across the world were obtained from the USDA-ARS, Germplasm Resource Information Network, Plant Genetic Resources Conservation Unit, Griffin, GA and World Vegetable Center  (Table C. annuum collection was comprised of 90 domesticated cultivars and 4 wild accessions. The C. baccatum collection had 218 lines of C. baccatum var. pendulum and 17 wild accessions (C. baccatum var. baccatum). Peduncle length (cm) was measured for 5 plants each of 217 accessions belonging to C. baccatum var. pendulum grown in a greenhouse in three replications.A representative sample of 377 pepper accessions (283 n) Table . The C. https://bitbucket.org/tasseladmin/tassel-5-source/wiki/Tassel5GBSv2Pipeline). Chromosomal assignment and position on the physical map of various SNPs were deduced from the C. annuum whole genome sequence (WGS) draft at http://peppergenome.snu.ac.kr. SNPs were designated by chromosome number and position .Genomic DNA was isolated from the seedlings using the DNeasy plant mini kit , and GBS was as described . Observed nucleotide diversity (π) and Tajima's D were estimated by using TASSEL v5.0 with a sliding-window approach as described  was estimated on the basis of the Wright F statistic  with the SNP & Variation Suite (SVS v8.1.5)  algorithm  and the PCA correction technique; the method of stratification was as described  analysis  and domesticated (C. annuum + C. baccatum) accessions was 0.09 and 0.05, respectively. The FSTbetween domesticated C. annuum and C. baccatum was 0.78, which indicates genome-wide divergence. The FST between wild C. baccatum and wild C. annuum was 0.66. Crown-shaped fruited C. baccatum types are unique for this species group, and pairwise FST values with wild, semi-domesticated and domesticated were 0.10, 0.06, and 0.03, respectively, which indicates their closeness to domesticated types. FST-values for semi-domesticated with wild and domesticated C. baccatum types were 0.03 and 0.01, respectively. We present an overall FST distribution in a Manhattan plot for all chromosomes showing important chromosomal regions with the highest FST as peaks  and Tajima's D across various chromosomes to understand genome-wide bottleneck effects. The frequency of segregating SNPs as reflected by various chromosomal measures of mean π and Tajima's D is presented in Figure C. baccatum, chromosome 4 was positive for π and Tajima's D which indicates accumulation of rapid mutations on this chromosome. The remaining chromosomes were negative or nearly negative for Tajima's D, which indicates bottlenecks in domestication. In contrast, C. annuum chromosomes were positive for Tajima's D on all chromosomes except chromosome 8, which indicates differential evolution after the domestication or the influence of diverse breeding.Because of the strong population structure, we assessed patterns of variation separately for each group of domesticated accessions from the respective species when making inferences about the evolutionary dynamics of domestication. Crop domestication is often associated with “population bottlenecks” because of the limited number of founding individuals experiencing domestication events. These bottlenecks may be evident in pepper when comparing diversity between cultivated forms of C. baccatum accessions on all adjacent marker pairs within a chromosome or within a haplotype block. Haplotype distribution is important to understand patterns of genetic variation of C. baccatum gene pools and has a wide range of applications. The 2 major processes that shape haplotype structure are the domestication process and breeding history. We used “minimize historical recombination,” a block-defining algorithm developed by Gabriel et al. (r2) and LD estimate (D′) for the EM and CHM methods are in Table r2 and we compared the pattern of decay at different levels. With pair-wise analysis considering adjacent SNPs across chromosomes, most SNP associations were within 50 kb  in cultivated C. baccatum indicates a population bottleneck during domestication or through the breeding histories, or the speciation of C. baccatum might have occurred with relatively narrow genetic diversity. In contrast, C. annuum chromosomes showed positive Tajima's D on all chromosomes except chromosome 8, which indicates that speciation or domestication of C. annuum might have occurred at multiple sites, contributing to wider genetic diversity as discussed by Kraft et al. , plays an integral role in GWAS and determines the density of SNPs required for GWAS , serine/threonine protein kinase, ABC transporter gene and RING finger protein, which may play important roles in growth and development as well as cell wall integrity and elongation as has been shown in other plants (Lally et al.,  Bulmer, .C. annuum and C. baccatum. Mapping all the SNPs to the C. annuum reference genome helped to identify homologous SNPs between the two incompatible cultivated pepper genomes, which was further useful to reduce ascertainment bias, so this SNP set was useful in estimating genome-wide population differentiation and allele sharing between the two genomes. Furthermore, the SNPs anchored to the C. annuum genome may not be in the same order in the C. baccatum genome because some genomic regions may not be co-linear to the C. annuum genome because of genome rearrangements. In a comparison of C. baccatum and C. annuum linkage maps, Lee et al. (Our study describes the utility of SNPs generated by GBS for genome-wide divergence and LD patterns between e et al.  identifiC. baccatum genome. This SNP panel and the results pertaining to population structure, IBS and LD decay analyses will facilitate routine use of GWAS for identification of genes associated with various economically important traits in Peruvian peppers. Our identification of SNPs associated with fruit peduncle length demonstrates opportunities for utilization of GWAS in crop improvement.Such uncertain positions of SNPs can be corrected only when the whole genomesequence is available for UR, PN, JS, GH, and AE designed the study and drafted the manuscript. PN, VA, JD, and BD conducted peduncle phenotyping. PN, VA, AA, JD, and BD extracted DNA and assisted to generate genome-wide SNPs. DC provided whole genome sequence draft and mapped SNPs to the genome. UR, PN, CR, TS, AA, and VA performed population structure and GWAS analysis.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.


Marssonina leaf spot disease of poplar (MLDP), caused by the hemibiotrophic pathogen Marssonina brunnea, frequently results in damage to many poplar species. In nature, two formae speciales of M. brunnea exist that are susceptible to different poplar subgenera. Marssonina brunnea f. sp. monogermtubi infects poplar hosts from Populus sect. Aigeiros (Aig), while M. brunnea f. sp. multigermtubi always infects poplar hosts from Populus sect. Leuce Duby (Leu). Based on the fungal penetration structures, a comprehensive transcriptomic approach was used to investigate the gene expression patterns of these two poplar subgenera at three crucial infection stages. MLDP significantly altered the expression patterns of many genes involved in mitogen activated protein kinase (MAPKs) and calcium signaling, transcription factors, primary and secondary metabolism, and other processes in both poplar subgenera. However, major differences in gene expression were also observed between the two poplar subgenera. Aig was most responsive at the initial infection stage, while Leu largely interacted with M. brunnea at the necrotrophic phase. Furthermore, the differentially expressed genes (DEGs) involved in pathways related to biotic stress also differed substantially between the two poplar subgenera. Further analysis indicated that the genes involved in cell wall metabolism and phenylpropanoid metabolism were differentially expressed in the progression of the disease. By examining the expression patterns of genes related to the defense against disease, we found that several genes annotated with causing hypersensitive cell death were upregulated at the necrotrophic phase of MLDP, inferring that plant immune response potentially happened at this infection stage. The present research elucidated the potential molecular differences between the two susceptible interaction systems in MLDP and provided novel insight into the temporal regulation of genes during the susceptible response. To the best of our knowledge, this study also constitutes the first to reveal the molecular mechanisms of poplar in response to the transition of hemibiotrophic fungal pathogens from the biotrophic phase to the necrotrophic phase. Plant–pathogen interaction networks are complex, and long-term co-evolution has resulted in the establishment of two primary means of communication between plants and their pathogens. Plants attempt to recognize pathogens and defend themselves against potential pathogens on their surface, while pathogens, in contrast, endeavor to manipulate the biology of the plant to complete their growth and reproduction . UnderstPopulus spp.) are important commercial tree species that are globally distributed and have become the model trees for forests [Populus comprises six sections: Abaso, Leuce, Leucoides, Aigeiros, Turanga, and Tacamahaca [Marssonina leaf disease of poplar (MLDP), which mostly causes early defoliation, is one of the main diseases affecting poplar [Marssonina spp. on the young leaves [Marssonina brunnea [Populus deltoids lipoxygenase genes (PdLOX1 and PdLOX2), and two extracellular proteins, PdPGIP2 and PdPGIP4, were both upregulated under the penetration of M. brunnea [PtrWRKY genes contained in the complete WRKY domain in Populus spp. were also induced during MLDP development [Poplars , while M. brunnea f. sp. multigermtubi always infects poplar hosts from Populus sect. Leuce Duby (Leu) [M. brunnea towards Aig and Leu implies complex interactions between plants and pathogens. Transcriptome analysis of M. brunnea f. sp. multigermtubi on the leaves of resistant and susceptible poplar has been conducted [M. brunnea is a typical hemibiotrophic pathogen [M. brunnea f. sp. multigermtubi. An assessment of the interactions between M. brunnea f. sp. monogermtubi and Leu is still lacking and limits the understanding of the differences between the MLDPs caused by the two formae speciales. Additionally, it is worth noting that previous studies were all designed and analyzed without considering the concrete microscopic histopathology and hemibiotrophic traits of M. brunnea. Additional research is required to systematically recognize and analyze MLDP more accurately.In China, MLDP is mainly caused by ateaceae . Reportsgermtubi ,18,19. Iby (Leu) ,19. It iby (Leu) ,18,19. Tonducted . In prevpathogen . All theM. brunnea in poplar; and (iii) to elucidate the potential different molecular response mechanisms of the two poplar sections to MLDP. To achieve this, RNA-Seq analysis was used to analyze the transcription profiles of 31,952 poplar genes and global patterns in poplar gene expression within two types of MLDP, M. brunnea f. sp. multigermtubi infecting Aig, and M. brunnea f. sp. monogermtubi infecting Leu, following three important infection stages. This enabled the identification of significantly differentially expressed genes (DEGs) during disease development and revealed the major metabolic processes and signal pathways in poplar associated with the development of MLDP. These results are important for understanding the molecular mechanisms of MLDP pathogenesis and indirectly inform breeding programs.The aims of the current study were three-fold: (i) to identify poplar genes that are differentially expressed in response to MLDP in two poplar sections; (ii) to explore crucial genes associated with the development of MLDP and further analyze the physiological and biochemical changes caused by pathogenic infection of Populus deltoids cv. Zj-2 clones belonging to Aig, and Populus alba × Populus alba var. pyramidalis clones belonging to Leu. They were both obtained from the poplar seedling station of the Poplar Experimental Bureau . Annual poplar cuttings were cultured in a glasshouse maintained at 25 ± 2 °C under a relative humidity (RH) of 50–55% during the day, and at 22 ± 2 °C under 55–65% RH at night. Light during the day was supplied from 08:00 to 20:00 by sunlight lamps. The studied fungi were M. brunnea f. sp. monogermtubi (MO45) isolated from Populus tomentosa in Beijing, and M. brunnea f. sp. multigermtubi (MU39) isolated from Populus nigra . During isolation, the infected leaves were first washed by sterile water, sterilized about 30 s in 75% ethanol, and then kept in moist conditions for 2 days at 25 °C for developing fruit bodies. When the white conidia were produced, they were transferred onto potato-dextrose agar (PDA) containing 50 µg/mL streptomycin sulfate using an inoculating needle. Finally, the isolates were cultured on PDA medium at 25 °C in the dark and then stored in tubes filled with PDA inclined medium at 4 °C. The cultivars of poplar used were The strain method was used to visualize the MLDP infection process by microscopy. Briefly, the inoculated leaves were collected and cut into 1 × 1 cm pieces. After de-coloring for 12 h in 0.15% saturated trichloroacetic acid bleaching liquid , the samples were transferred into a mixed stain solution ) and stained for nearly 4 h at 58 °C. The samples were then observed under a light microscope .5 conidia/mL. Fully expanded healthy leaves were collected from the poplar seedlings, sterilized in 75% ethanol for 20 s, washed with sterile water three times, and then placed on wet filter paper upon a layer of water agar medium in Petri dishes. The spore suspension was sprayed onto the surface of the leaves, and the inoculated leaves were incubated in a manual climatic incubator at 25 °C and 100% RH under a 12 h photoperiod (08:00 to 20:00) using sunlight lamps. For observation, the inoculated leaves were harvested at six h post inoculation (hpi), 36 hpi, and 96 hpi. In the control group (CK), the leaves were sprayed with distilled water and harvested at six hpi. The samples were then immediately frozen in liquid nitrogen and stored at −80 °C for RNA isolation. Inoculations on detached leaves have proven to be available for the research of MLDP [The inoculum was obtained by washing the conidia from the incubated leaves with sterile distilled water and then adjusting the concentrations to approximately 5 × 10 of MLDP ,20,22.The RNA extraction of the samples used TRIzol reagent  according to the instruction manual. The quality of all RNA was confirmed by agarose gel electrophoresis and determined with a NanoDrop 2000 spectrophotometer . The mRNA was isolated by NEBNext Poly (A) mRNA magnetic isolation module . The complementary DNA (cDNA) library was constructed following the manufacturer instructions of the NEBNext ultra RNA library prep kit for Illumina (New England Biolabs) and NEBNext Multiplex Oligos for Illumina (New England Biolabs). Briefly, the enriched mRNA was fragmented into approximately 200 nt RNA inserts, which were used to synthesize the first-strand cDNA and the second-strand cDNA. The double-stranded cDNA was synthesized via end-repair/dAdenosine-tail and adaptor ligation. The suitable fragments were isolated using Agencourt AMPure XP beads  and enriched by PCR amplification. Finally, the constructed cDNA libraries were sequenced on a flow cell using an Illumina HiSeq™ 2500 sequencing platform . The RNA-seq data are available from the National Center for Biotechnology Information (NCBI) database with the BioProject number PRJNA397718. Populus trichocarpa, downloaded from Phytozome [Low quality reads were filtered by excluding those with only adaptors, and unknown nucleotides were removed. The clean reads filtered from the raw reads were mapped to the v3.0 genome of hytozome  using Tohytozome  with somhytozome , which wp-value less than 0.001 and a threshold fold change (log2) ≥ 2 was used to call differentially expressed genes between two samples. The identification of unique or overlapping genes within the different expression data of the samples and the generation of Venn diagrams were constructed using Draw Venn Diagram [For the two poplar sections, DEGs were analyzed using DESeq2  in R pac Diagram . Heat ma Diagram . Common  Diagram  using thP. trichocarpa as the reference set. The GO enrichment was completed under a significance threshold of 0.05 following Hochberg FDR correction. The enriched GO terms were then summarized using the online analysis tool REVIGO [clusterProfile [Pathview [Gene Ontology (GO) term annotation and GO enrichment for poplar genes were obtained by online analysis on agriGO version 2.0  with P. l REVIGO . Kyoto El REVIGO  package rProfile  and visuPathview . AdditioPathview .RNA samples were firstly purified with DNase I (RNase-free) . Quantitative reverse-transcription (qRT)-PCR was performed with Oligo-DT and SuperScript III reverse transcriptase (Invitrogen). All qRT-PCR reactions were performed with SuperReal Premix Plus  and carried out on an ABI 7500 real-time PCR system . Relative expression levels were calculated using the ΔΔCT method , with 60The susceptible and resistant systems of MLDP were identified based on the inoculation experiment. Specifically, Aig was susceptible to MU39 and resistant to MO45, while Leu was susceptible to MO45 and resistant to MU39 a. HistopP. trichocarpa ranged from 50.58% to 73.55%  value of the samples all exceeded 85%. The percentage of mapped reads across all samples to the genome of o 73.55% . Among to 73.55% . The coro 73.55% a. A simio 73.55% b. Replico 73.55% b. The dio 73.55% b.From 28,520 to 29,561 genes were expressed at the level (0 < FPKM) in Aig, while from 28,915 to 29,713 genes were noted in Leu c. In genTo validate the expression patterns identified by the RNA-Seq, 18 common genes with different expression levels were selected to perform qRT-PCR analysis for Aig and Leu . AnalysiM. brunnea  was only enriched at 96 hpi , cell wall organization or biogenesis (GO:0071554), lipid metabolic processes (GO:0006629), microtubule-based movement (GO:0007018), protein ubiquitination (GO:0016567), antioxidant activity (GO:0016209), microtubule motor activity (GO:0003777), oxidoreductase activity (GO:0016491), transferase activity transferring glycosyl groups (GO:0016757), and other terms . Furthert 96 hpi . Additioresponse to external stress was also enriched at 96 hpi in Leu , Potri.002G082400 (ILL6), and Potri.002G234000 (ATB2) were downregulated at six hpi (ATPIN1/PIN1) and Potri.013G144300 (DFL2), were significantly upregulated at six hpi and 36 hpi, and then downregulated at 96 hpi  was significantly downregulated at six hpi  annotated with “cytochrome P450 superfamily protein”, and two genes (Potri.001G386900 and Potri.005G126400) annotated related to “brassinosteroid signaling positive regulator” remained downregulated (UGT74E2), involved in the salicylic acid (SA) signaling pathway, was always significantly downregulated (abscisic acid (ABA) signaling pathway, was significantly upregulated at six hpi and 36 hpi, and then significantly downregulated at 96 hpi , including genes related to pectin metabolism, cellulose metabolism, and saccharide metabolism, were all upregulated during the three infection stages (ARA1), Potri.001G136200 (CSLD3), Potri.008G153000 (frigida-like protein), Potri.010G109400 (plant invertase/pectin methylesterase inhibitor superfamily), Potri.018G094800 (XTH22), and three other genes encoding expansins , remained downregulated during the disease progression  were sharply downregulated at six hpi and significantly upregulated at 96 hpi , including Potri.012G043200 (MKK9) and Potri.001G400500 (RLP33), remained downregulated as the disease progressed and were significantly reduced at 96 hpi  showed no expressional changes in the initial two infection stages, but was sharply upregulated at 96 hpi. Additionally, four genes in this class, namely Potri.001G017600, Potri.001G079900 (ERF-1), Potri.001G079600, and Potri.001G048200 remained downregulated with a decreasing tendency as the disease progressed  remained upregulated as the disease developed  family proteins remained upregulated as the disease progressed  TFs, and WRKY TFs were mostly upregulated (DNA-binding one zinc finger (DOF), Respiratory burst, and SA had no common genes , three genes encoding 4-coumarate-CoA ligase (4CL), one gene encoding cinnamoyl-CoA reductase (CCR), one gene encoding cinnamate 4-hydroxylase (C4H), three genes encoding caffeic acid 3-O-methyltransferase (COMT), and four genes encoding peroxidases (CCR (Potri.003G181400) and C4H (Potri.019G130700) were highest , CHI (Potri.010G213000), F3M (Potri.013G073300), and LDOX (Potri.001G113100) exhibited the highest expression levels  and downregulated expression from the biotrophic stages to the necrotrophic stage (96 hpi) . As M. bactivity . The KEGactivity . Within  dextrin . Phenylpoxidases . Among t highest . The gen two LAR . Of thesn levels .NOP1 (Potri.015G147500), NOP56 (Potri.012G092900), SNU13 (Potri.013G116800), and NOP10 (Potri.005G226300) maintained higher expression values compared to the other genes , 24 of which were classified in generation of precursor metabolites and energy . Eleven er genes . Furtherer genes .M. brunnea infection, including Potri.013G097000, Potri.017G133600, Potri.001G379700, Potri.006G195300, and Potri.007G039300, which were most highly expressed at 96 hpi  exhibited the highest expression levels, indicated upregulated expression as the disease progressed a. Seventt 96 hpi b. Twentyt 96 hpi b. Elevent 96 hpi b. Many dt 96 hpi c.CRK10), Potri.014G147300, Potri.019G099200, Potri.T090500 (CRK25), and Potri.003G166100 (SERK2) were most highly expressed , Potri.013G059900 (RLK1), Potri.017G094400 (RLK2), Potri.014G144600 (RLK2), and Potri.013G036600 , Potri.012G124100, Potri.004G024900 (CRK25), Potri.018G111700 (CRK34), and Potri.004G023800 (CRK10) exhibited the largest difference between six hpi and 96 hpi (Receptor-like protein kinases (RLKs) are transmembrane proteins that contain putative amino-terminal extracellular domains and carboxyl-terminal intracellular kinase domains, and participate in many biological processes, including plant disease resistance . For Aigxpressed . Sevente3G036600 . In Leu,d 96 hpi . There wd 96 hpi . The majd 96 hpi .M. brunnea and exhibited a downregulated trend as the disease progressed  were obtained in comparison to existing genomic transcriptome studies in MLDP (27.3 GB) . To furtM. brunnea is a typical hemibiotrophic pathogen, these NBS-LRR proteins might have important functions for poplars in MLDP resistance. We identified 145 and 141 genes containing NBS-LRR domains in Aig and Leu respectively, and of these genes, 16 and 13 were respectively associated with Aig and Leu -triggered immunity (PTI) and effector-triggered immunity (ETI) . RLKs ar and Leu . Additiothaliana , was relMPK20, MPKK9, and MAPKKK18 in MAPK pathways exhibited differential expression as the disease progressed , MPK7 (Potri.007G020100), MPKK9 (Potri.015G030700), MPK3 (Potri.009G066100), MAPKKK18 (Potri.014G155000), and MAPKKK5 (Potri.002G129100) were differentially expressed  and most of them were upregulated when infected by M. brunnea. CDPKs can phosphorylate special substrates in PTI and ETI to control transcriptional reprogramming of immune genes and the hypersensitive response [CAM8 (Potri.005G052800), which maintained downregulated expression following infection by M. brunnea, constitutes a uniquely differentially expressed CAM gene. The downregulation of pathogens induced by CAM was previously found to impact disease resistance in tobacco [A. thaliana CML were all upregulated during disease development , was always significantly downregulated during disease progression in both Aig and Leu (Phytopathogens have been reported to influence the primary metabolism of plants to achieve successful infection ,76. Path for Aig . Most do for Aig . Many ge for Aig . These r for Aig . These ranalysis . It is w and Leu . PreviouM. brunnea, a few genes involved in phenylpropanoid biosynthesis were upregulated in both poplars. Additionally, 13 genes, which were upregulated at the biotrophic stages and downregulated at the necrotrophic stage, were also enriched in phenylpropanoid biosynthesis pathways in Aig (M. brunnea expansion observed between two poplars. The cell walls of plants constitute an early defense against fungal penetration. Several upregulated DEGs annotated with pectin methylesterase inhibitors were enriched in pectinesterase activity in Aig; however, these genes in Leu were mostly downregulated at 96 hpi (M. brunnea, reinforcing the cell walls might limit the expansion of the pathogen. However, further studies are required to verify this. Flavonoids are also synthesized via the phenylpropanoid pathway and play important roles in the interactions between plants and other organisms [Many studies have shown that plants are able to synthesize and release secondary metabolites to protect against a wide variety of microorganisms . In resps in Aig . Generals in Aig . The dist 96 hpi . Pectin rganisms . In Aig M. brunnea triggered many changes in gene expression and altered the primary and secondary metabolism of the host cells in both two poplar sections. For poplars, the genes in MAPK signaling and calcium signaling were closely related with the reactions caused by the infection of M. brunnea. The genes associated with cell wall metabolism, photosynthesis, and phenylpropanoid metabolism played important roles for poplar in response to MLDP. Besides, hypersensitive responses probably happen during the development of MLDP. Furthermore, many important genes showed different expression patterns between the two poplars.In summary, M. brunnea at the necrotrophic phase. Our findings bring a novel resource for understanding the interactions between M. brunnea and poplar. There could be quantitative differences in virulence and in turn qualitative differences in gene expression between poplar species from the same section. It is worth mentioning that our results could not completely represent all poplar species of the two poplar sections. To completely understand recognitions on the level of poplar sections, more poplars species should be studied. To the best of our knowledge, this study also constitutes the first to reveal the molecular mechanisms of poplar in response to the transition of hemibiotrophic fungal pathogens from the biotrophic phase to the necrotrophic phase. Further investigations will be focused on functional mapping and validation of the crucial genes. In practice, this study provides useful information for guiding the development of poplar resistant varieties toward M. brunnea.This study presents a large set of DEGs and elucidated their temporal regulation during specific stages of disease development. These data are important for understanding the molecular mechanism of the development of this disease. By comparing two different susceptible interaction systems of this disease, we provide useful resources for revealing the molecular mechanisms of this complicated phenomenon. Many genes related to cell wall metabolism and phenylpropanoid metabolism present similar expression trends with the disease development between two poplars. However, major differences in gene expression were also observed between the two poplar subgenera. Aig was most responsive at the initial infection stage, while Leu largely interacted with


Rhinocladiella mackenziei accounts for the majority of fungal brain infections in the Middle East, and is restricted to the arid climate zone between Saudi Arabia and Pakistan. Neurotropic dissemination caused by this fungus has been reported in immunocompromised, but also immunocompetent individuals. If untreated, the infection is fatal. Outside of humans, the environmental niche of R. mackenziei is unknown, and the fungus has been only cultured from brain biopsies. In this paper, we describe the whole-genome resequencing of two R. mackenziei strains from patients in Saudi Arabia and Qatar. We assessed intraspecies variation and genetic signatures to uncover the genomic basis of the pathogenesis, and potential niche adaptations. We found that the duplicated genes  are more susceptible to accumulating significant mutations. Comparative genomics with other filamentous ascomycetes revealed a diverse arsenal of genes likely engaged in pathogenicity, such as the degradation of aromatic compounds and iron acquisition. In addition, intracellular accumulation of trehalose and choline suggests possible adaptations to the conditions of an arid climate region. Specifically, protein family contractions were found, including short-chain dehydrogenase/reductase SDR, the cytochrome P450 (CYP) (E-class), and the G-protein β WD-40 repeat. Gene composition and metabolic potential indicate extremotolerance and hydrocarbon assimilation, suggesting a possible environmental habitat of oil-polluted desert soil. Rhinocladiella is a polyphyletic genus residing in the fungal family Herpotrichiellaceae , comprising a number of opportunistic pathogens on humans . Cases reported outside the endemic area, e.g., in Europe or the U.S., have invariably concerned immigrants from countries in the Middle East  and aldehyde dehydrogenases (ALDHs), membrane transport proteins, and a diverse ensemble of CYPs, which are believed to be essential for extremotolerance and survival of environmental stress conditions , pH 8.0 in 2 ml screw-capped tubes, and then resuspended in 500 µl TE buffer. Fungal cell walls were disrupted using 0.5 mm glass beads in a BioSpec Mini-Beadbeater-16 (BioSpec) for 5 min and cooled on ice for an additional 5 min. DNA solutions were separated using two phenol/chloroform  extractions. DNA was then precipitated by isopropanol, washed with 70% ethanol, dried at room temperature, and resuspended in 35 µl TE buffer, pH 8.0. DNA quantity and quality were determined using Qubit , and an Agilent Bio Analyzer 2100 using a 1000 DNA Chip (Agilent).To extract genomic DNA, fungal mycelia of the http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc) and low-quality sequences were removed by Trimmomatic  . The libraries were validated and quantified without bead normalization using an Agilent Bio Analyzer 2100 1000 DNA Chip (Agilent). The dH24460 and IHM 22877 genomes were sequenced on the Illumina MiSeq platform using a paired-ends protocol and a version-3 600 cycles kit. Quality control was performed using FastQC v0.11.3 (R. mackenziei CBS 650.93 (accession JYBU00000000) by using Burrows Wheeler Aligner (BWA) v0.7.5 mem (https://software.broadinstitute.org/gatk/). SNP annotation was performed by VCFannotator (http://vcfannotator.sourceforge.net/) to assess whether the SNP was found within an untranslated region, intron, or coding exon. Mutations were classified into synonymous (SYN), nonsynonymous (NSY), read-through (RTH), and nonsense (STP). We used the alignment-based method PROVEAN to predict the effect of NSY SNPs on protein function  were aligned to the genome of the strains IHM 22877 and dH24460 using the homology-based predictor Exonerate v. 2.2.0 . Orthologs and paralogs were determined by means of OrthoMCL  from the raw sequencing data. The bait used for the GRAbB assembly was the mitochondrial genome of E. dermatitidis (NW_008751656). Initial mitochondrial genome annotations were done using MFannot  and manually adjusted. Annotation of tRNA genes was improved using tRNAscan-SE  and GCA_001723235 (strain dH24460). Raw sequence data can be accessed through the NCBI Sequence Read Archive: accession number SRP128022. R. mackenziei strains from Qatar and Saudi Arabia compared to the reference genome, each strain was deeply sequenced using Illumina MiSeq. After filtering, a total of 12,996,368 and 10,131,024 high-quality reads were generated for the strains dH24460 and IHM22877, respectively. Among these reads, 11,542,511 and 9,568,960 reads were mapped to the R. mackenziei CBS 650.93 reference genome (To characterize variants of two e genome .http://transposonpsi.sourceforge.net) to identify potential transposable elements. The scaffold harbored three putative LTR retroelements: one classified as Ty1/Copia and two classified as gypsy.Sequencing of both dH24460 and IHM 22877 strains did not produce reads corresponding to the region of scaffold 15 in the reference genome. This scaffold was 4775 bp long and contained two protein-coding genes (Z518_11416 and Z518_11417) annotated as hypothetical proteins, holding PFAM functional domains associated with LTR retrotransposons (PF03732)/zinc finger domains (PF00098) and manipulation of chromatin (PF00385). In order to further investigate the genomic composition of scaffold 15, we used TransposonPSI  for strain dH24460 and in scaffold 3 (one every 484 bp on average) for strain IHM 22877. The distribution of SNPs along the genomes is shown in Materials and Methods) was used to classify the mutations into four distinctive categories: SYN, NSY, RTH, and STP. Variant annotation showed that 78,041 SNPs (54.4%) were located in intergenic regions , while 65,291 SNPs (45.6%) were located in intronic  and exonic  regions. Of the total SNPs localized in coding regions, 31,409 were SYN , 26,700 were NSY , 86 were RTH , and 394 were STP   .Materials and Methods). Among this gene set, 325 and 422 intronic SNPs, and 5287 (3258 SYN and 2024 NSY) and 6547 (2577 SYN and 3964 NSY) exonic SNPs, were identified in the strains IHM 22877 and dH24460, respectively. SNPs within SCO exons included only four STP (1%) and RTH mutations of stop codons (8%)  that were common to 22 black yeasts (see ons (8%) . STP mutTable S2). The proteins carrying these mutations, in both strains, were enriched for GO categories involved in oxidoreductase activity (P < 2.2 × 10−16), which may contribute to diversification of these functions. Among the 872 putative paralogous genes predicted in R. mackenziei, 186 carry at least one severe mutation.Among the NSY SNPs, 1266 (8.5%) and 1011 (8.3%) mutations in strains dH24460 and IHM 22877, respectively, were considered to have significant effects on protein function . Intronic regions comprise 5.8% of the indels (dH24460: 845 and IHM 22877: 722). Rates of SNPs and indels were more frequent in R. mackenziei dH24460, revealing that this is more highly diverged from the reference isolate.We identified 26,754 potential indels . Overall, 7.7% of these mutations were found in coding regions with a mean length of 7.3 and 8.4 bp for insertions, and 14.9 and 16.6 bp for deletions, in the strains dH24460 and IHM 22877, respectively. Indels that are multiples of 3 bp, and thus cannot cause frameshifts, account for 42% of the coding indels in dH24460 and 38% in IHM 22877  and dH24460 (GenBank assembly accession: GCA_001723235). The reference protein set contained 11,382 proteins. In total, 11,148 genes were predicted in strain IHM 22877 and 11,191 genes in dH24460, covering 97.9 and 98.2% of the gene content of the type strain, respectively. Using the reference protein set, a total of 6349 proteins (55.8%) were assigned to GO terms and 8836 proteins (77.5%) contained a protein family domain. The top five most abundant PFAM domains included WD40 repeat PF00400, membrane transport proteins major facilitator type (MFS) PF07690, fungal transcription factor PF04082, transcription factor type Zn2Cys6 PF00172, and CYP PF00067. Comparative analyses with other black yeasts showed that the R. mackenziei genomes contained a large number of highly conserved homologs. Best-hits BLASTP (cut-off identity > 90%) were found against the saprobic species E. xenobiotica (10.3%), F. multimorphosa (9.7%), and C. immunda (8.6%), followed by two neurotropic fungi, E. dermatitidis (8.4%) and C. bantiana (8.0%). The lowest similarities were found with P. attae (0.5%) and P. europaea (0.4%), members of black yeast family Cyphellophoraceae. Interestingly, highly conserved homologs shared between the neurotropic fungi R. mackenziei, C. bantiana, and E. dermatitidis, spanned across several GO categories (Table S3 and Table S4), including organonitrogen compound biosynthetic/metabolic process , amide biosynthetic process (GO:0043604), and peptide metabolic process (GO:0006518).Protein-coding genes were predicted based on alignments of proteins of R. mackenziei and other 22 black yeast species. This analysis indicated the presence of 872 putative paralogs in R. mackenziei. These genes belong to the same GO categories described above. Additional mechanisms involving transmembrane transport, mainly of nitrogen, were found overrepresented in R. mackenziei, such as nitrogen transport (GO:0071705), amino acid transport , peptide transport (GO:0015833), amide transport (GO:0042886), and amino sugar and aminoglycan biosynthetic processes (GO:0046349) (Table S5). These findings raise new questions regarding the potential involvement of nitrogen-containing compounds and neurotropism observed in some black yeast species.Orthology analysis using Orthomcl determined the protein families conserved in R. mackenziei possesses 755 orphan proteins that are unique among the black yeasts according to Orthomcl , and therefore were not included in any group of homologs (Table S6). InterProScan searches performed on this data set showed that 241 proteins possess conserved functional domains. The top five most frequent Interpro protein domains (IPRs) in orphan proteins include IPR020683 (ankyrin repeat-containing domain), IPR001128 (CYP), IPR011701 (MFS transporters), IPR010730 (heterokaryon incompatibility), and IPR002347 (short-chain dehydrogenase/reductase). Despite the low sequence conservation of these proteins among black yeasts, BlastP analysis against the NCBI database revealed that many of these enzymes are often found in plant-associated fungi (Table S6), for example the putative glutathione S-transferase protein Z518_06694 (86% identity with Eutypa lata), nitrilase Z518_09980 (66% identity with Botrytis cinerea), CYP Z518_01777 (93% identity with Oidiodendron maius), the putative siderophore iron transporter Z518_06150 (identity 77% with Phaeomoniella chlamydospora), and the myo-inositol transporter Z518_03615 (74% with O. maius). Other enzymes involved in the iron transport, such as the siderophore iron transporters Z518_10557 and Z518_10557, were similar to the saprobic fungus Purpureocillium lilacinum.R. mackenziei, the pool of secreted proteins (secretome) includes several virulence factors, such as proteases and chitinases. Analyses of secretion signal peptides, the N-terminal regions that control the entry of proteins to the secretory pathway, revealed the presence of 216 putative secretory proteins in this fungus. Among these enzymes, 20 peptidases, 46 carbohydrate-active enzymes (CAZY), four laccases, and one CYP were identified (Table S7). The signal peptide was also found in three out of four tyrosinases reported in R. mackenziei. Interestingly, extracellular tyrosinases and laccases are involved in oxidation and scavenging of phenolic compounds, as well as in the melanin biosynthesis  and following the International P450 Nomenclature Committee rule, which suggests that proteins with > 40% identity and > 55% identity may be clustered under the same family and subfamily, respectively. For that, we used phylogenetic analysis including 645 CYP protein sequences from the black yeasts C. bantiana, C. psammophila, E. dermatitidis, E. xenobiotica, and R. mackenziei (Table S8). Three CYPs belonged to the CYP52 family, which is involved in alkane assimilation and fatty acid metabolism in yeasts  and malate synthase (Z518_01715). Similarly to other black yeast species, R. mackenziei possessed a paralog copy for isocitrate lyase. glucose . This pa glucose . Two enzR. mackenziei CBS 650.93 was assessed in order to identify genes linked to the degradation of aromatic compounds, such as benzene, toluene, ethylbenzene, xylene, and styrene, important pollutants from petrochemical and chemical industries. Toluene is initially oxidized to benzyl alcohol by a membrane-bound CYP in toluene-growing cells of the closely related C. saturnica CBS 114326, previously confused with Cladosporium sphaerospermum , a CYP belonging to the family CYP504. Homogentisate is oxidized to 4-maleylacetoacetate by the homogentisate 1,2-dioxygenase , which is converted to fumarylacetoacetate and to fumarate/acetoacetate by the enzymes maleylacetoacetate isomerase (Z518_00072) and fumarylacetoacetase (Z518_00072), respectively. This data suggests that the catabolism of aromatic amino acids, such as phenylalanine, can occur via oxidation to phenylacetate. Previously studies suggested that a CYP504 family member is involved in the catabolism of both phenylacetate and 2-hydroxyphenylacetate acid, which is then oxidized to homogentisate and degraded to fumarate and acetoacetate  in E. dermatitidis were also found in R. mackenziei. In contrast to other neurotropic fungi  are adjacent in R. mackenziei. The SidA and SidC genes  are adjacent in both R. mackenziei and E. dermatitidis. The cluster containing the high-affinity iron permease FtrA and the ferroxidase FetC are duplicated in R. mackenziei (Z518_02600- Z518_06281 and Z518_02599-Z518_06280), as well as in the neurotropic black fungi E. dermatitidis (HMPREF1120_01590-HMPREF1120_04510 and HMPREF1120_01589-HMPREF1120_04509) and C. bantiana (Z519_09337- Z519_11106 and Z519_09338-Z519_1110).Different strategies for iron acquisition were also present in R. mackenziei possesses distinctive mechanisms to enable its survival under extreme environmental conditions, such as the heat and the dryness of the desert where the environmental phase of the fungus is thought to reside. To avoid dehydration of cells, some fungi are able to accumulate neutral and low-molecular-weight compounds intracellularly, for example the disaccharide trehalose and the quaternary ammonium compound choline  that may be involved in extracellular choline uptake, which is subsequently converted to the osmoprotectant glycine betaine by the enzymes choline dehydrogenase (Z518_00519) and betaine-ALDH (Z518_07684). Among the black yeasts, homologs of choline permease are only found in the hydrocarbon-degrading fungus E. oligosperma (PV06_02302) and in the plant-associated fungus Ca. epimyces (A1O3_02156). In addition, we found that R. mackenziei harbors duplicated enzymes that catalyze the biosynthesis of trehalose through the processing of α-D-glucose-1P: the trehalose 6-phosphate synthase (Z518_04836 and Z518_10013) and the trehalose 6-phosphate phosphatase (Z518_02299 and Z518_05120). choline . R. mackR. mackenziei against closely related black yeast species , and members of the orders Eurotiales  and Onygenales . We confirmed the expansion of ADH-related domains (IPR013149 and IPR013154), CYP domains (IPR001128), and a trichothecene efflux pump domain (IPR010573) previously determined using a stochastic model of gene birth and death to estimate the evolution of gene family size (R. mackenziei and in other black yeasts: MFS transporters (IPR011701 and IPR020846) and sugar-associated transporters (IPR003663 and IPR005829), polyketide synthase (IPR020843), fungal transcription factor (IPR007219), and protein phosphorylation-related domains (Table S9).To investigate the gain and loss of protein families, we compared the repertoire of IPRs of R. mackenziei includes the glucose/ribitol dehydrogenase (IPR002347), the CYP E-class (IPR002401), and the G-protein β WD-40 repeat (IPR020472) (Table S9).The list of contractions in R. mackenziei. In general, the species shared many potential virulence-associated homologs with the neurotropic fungus Cryptococcus neoformans (Table S10). Sequence analyses revealed that gene Z518_10204, with equivalent copies found in all three R. mackenziei strains examined, corresponded to the DEAD-box RNA helicase VAD1  and is expressed during infection of human brain (Co. immitis (83% identity) and T. rubrum (83% identity), possessed homologs to Z518_10204. Additionally, gene Z518_03960, which encodes a phosphoenolpyruvate carboxykinase (PCK1) and is a virulence factor dependent on Vad1, was found conserved in R. mackenziei compared with Cr. neoformans (64% identity).In total, 520 pathogenicity-related genes were found in R. mackenziei and C. neoformans. This protein has been postulated as an important pathogenicity-related gene facilitating fungal central nervous system invasion , their amino-acid sequences are poorly conserved compared to C. neoformans. In contrast, Z518_09873, the R. mackenziei homolog of URE1, shares 62% of BLASTP identity with URE1 in C. neoformans. Accessory proteins are responsible for activating the urease apoenzyme and are thought to be involved in human brain infection  in R. mackenziei shares 88% of BLASTP identity with that in A. fumigatus. Gfa1 protein catalyzes the first step in the chitin biosynthesis pathway, and is believed to be essential and a potential drug target enzyme in A. fumigatus (R. mackenziei is AT-rich (∼26% G+C). It contains 14 conserved protein coding genes, two rRNA genes, and 25 tRNA genes, which code for all 20 amino acids , while IHM 22877 contains no introns. The introns are group I and contain homing endonuclease genes; two of them belong to the LAGLIDADG family and one to the GIY-YIG family (cob). Besides the intron difference there are minor (0.3%) sequence differences between the two strains. In total, 66 positions are variable, 24 of which are in intergenic and 42 are in coding sequences. The tRNA genes contain no variation, the rRNA genes contain 14 variable positions, and the protein-coding genes 28, but only five of them result in amino acid substitutions.Contrary to nuclear DNA, the circular mitochondrial genome of no acids . All gencob has its closest hit with an intron at the same position inside the cob gene of Fusarium oxysporum with 37% query coverage, 8e−98 E-value, and 75% sequence identity. BLAST search of the NCBI database returned no results for the other two introns.The intron inside R. mackenziei. Additionally, SVs were assessed, revealing an unexpected amount of large inversions. Hypervariable sections were seen in the ends of scaffolds (probably indicating telomeric regions) composed by repetitive nucleotides, which are subject to higher levels of mutations in other fungi . The notorious abundance of CYPs, ADHs, and ALDHs in the genomes of black yeasts is partially explained by gene duplication events, occurring in the common ancestor of these organisms, by which new genes with similar functions were copied , C. neoformans, and C. gattii [meningoencephalitis and propensity of C. gattii to form cryptococcomas , and epinephrine , in addition to the formation of melanin and neuromelanin (a brain-protective dark pigment synthesized from L-dopamine). Phenylacetate is also derived from environmental contaminants such as styrene and ethylbenzene , are also abundant in the mammalian central nervous system and might serve as an important carbon source for R. mackenziei . Overall, the pathway collection in this fungus resembles that of black yeast that are able to assimilate alkylbenzene hydrocarbons. These results confirm that, unlike other true pathogens, R. mackenziei is a versatile fungus, with several pathways to sequester carbon from a wide range of nutrient substrates from the environment, possibly suggesting an oligotrophic lifestyle. In this respect, the fungus differs from true pathogens that need to extract nutrients from their hosts. Opportunism may be explained by the occurrence of virulence factors that have other roles in the natural life cycle of the fungus, such as the presence of a glyoxylate cycle pathway, different strategies for acquiring iron, toxin neutralization, and secondary metabolism (e.g., in the phagolysosome during phagocytosis (Cr. neoformans (2 compounds via the glyoxylate shunt might also be a nutritional strategy adopted by oligotrophic black yeasts to survive in nutrient-depleted environments, avoiding loss of carbons as CO2 due to the decarboxylation steps in the tricarboxylic acid cycle.The evolution of true mammal pathogens, such as members of the order Onygenales (ocytosis . Howeverwww.g3journal.org/lookup/suppl/doi:10.1534/g3.117.300421/-/DC1.Supplemental material is available online at Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.


Strongyloides spp., gastrointestinal nematode parasites of humans and other animals, have genetically identical parasitic and free-living adult life cycle stages. This is an almost unique feature amongst nematodes and comparison of these two stages can provide insights into the genetic basis and evolution of Strongyloides nematode parasitism. Here, we present RNAseq data for S. venezuelensis, a parasite of rodents, and identify genes that are differentially expressed in parasitic and free-living life cycle stages. Comparison of these data with analogous RNAseq data for three other Strongyloides spp., has identified key protein-coding gene families with a putative role in parasitism including WAGO-like Argonautes (at the genus level) and speckle-type POZ-like coding genes (S. venezuelensis-S. papillosus phylogenetic subclade level). Diverse gene families are uniquely upregulated in the parasitic stage of all four Strongyloides species, including a distinct upregulation of genes encoding cytochrome P450 in S. venezuelensis, suggesting some diversification of the molecular tools used in the parasitic life cycle stage among individual species. Together, our results identify key gene families with a putative role in Strongyloides parasitism or features of the parasitic life cycle stage, and deepen our understanding of parasitism evolution among Strongyloides species. Strongyloides nematodes are soil-transmitted gastrointestinal parasites of humans and other animals. The Strongyloides life cycle has genetically identical adult parasitic female (PF) and free-living female (FLF) stages1, an almost unique feature amongst nematodes. The PF inhabits the mucosa of the small intestine of its host and produces genetically identical offspring by mitotic parthenogenesis. Eggs leave the host with the faeces and develop into infective larvae stage (iL3), which infect a new host via percutaneous penetration, either directly or via a free-living, dioecious adult generation. The free-living adult generation lives independently of a host and is not parasitic2. Because PF and FLF are genetically identical, differences between these two life cycle stages must be attributed to differences in transcriptional or post-transcriptional regulation of their genes. Direct comparisons between the transcriptomes of the PF and FLF can identify differentially expressed genes, enabling inferences to be made about the genes, and the proteins they code for, with a putative role in parasitism, or features associated with the parasitic life style3.Strongyloides species – S. ratti, S. stercoralis, S. venezuelensis and S. papillosus – and two closely related species, Parastrongyloides trichosuri (parasitic nematode) and Rhabditophanes sp. (free-living nematode) have recently been sequenced3. The genome sequences revealed they possess robust gene homology and synteny across species. Comparison of S. ratti and S. stercoralis RNAseq data for PF and FLF stages of the life cycle has uncovered key gene families with a putative role in Strongyloides parasitism4. Interestingly, many of the gene and protein families identified as having a putative role in Strongyloides parasitism, based on transcriptome and proteome evidence, are also reported to have a role in parasitism in a range of other parasitic nematode species7. Strongyloides spp. can therefore provide a suitable model system for studying the genetic and molecular basis of nematode parasitism, more generally.The genomes of four Strongyloides species can be phylogenetically grouped into two distinct subclades: S. venezuelensis-S. papillosus and S. ratti-S. stercoralis. These subclades can be characterised by the number of chromosomes they possess - S. ratti and S. stercoralis have two autosomes and one X chromosome; S. venezuelensis and S. papillosus possess two chromosomes , signifying some level of evolutionary divergence between these two subclades10. Different Strongyloides species have evolved to parasitise distinct and varied hosts; S. venezuelensis and S. ratti are parasites of rodents, S. stercoralis is a parasite of humans and dogs, and S. papillosus is a parasite of sheep. Specialism of Strongyloides species to their specific hosts indicates further evolutionary diversification within the Strongyloides nematode clade. Comparative analyses of the PF and FLF transcriptomes of these four Strongyloides species offers an interesting comparison of genes, and the proteins they code for, involved in parasitism, and can provide insights into the evolution of parasitism at the genus, subclade and species level. Here, we have performed RNAseq analysis of S. venezuelensis PF and FLF life cycle stages and compared these data with PF and FLF transcriptomes of three other Strongyloides species, to identify common and unique gene families putatively important in Strongyloides parasitism.The four sequenced S. venezuelensis life cycle, respectively . We compared these data to the genes that are differentially expressed across the analogous life cycle stages in three other Strongyloides species: S. papillosus   and lower for S. papillosus (1761 genes)  Fig. .Figure 1Strongyloides species, the differentially expressed genes belong to 3479 orthologue families , and among them 58 and 105 orthofamilies were commonly upregulated in all four species at the PF and FLF stages, respectively .Across all four 4  in S. ratti, S. stercoralis3 and S. papillosus11. In S. venezuelensis, astacin-coding genes were also the most commonly upregulated family in the PF transcriptome. However, only 2 SCP/TAPS-coding genes (of 159 SCP/TAPS-coding genes in the genome) were detected as upregulated in the S. venezuelensis PF transcriptome compared with 63, 64 and 102 genes  in the PF transcriptome of S. ratti, S. stercoralis and S. papillosus, respectively. Overall, the expression levels of S. venezuelensis SCP/TAPS-coding genes were higher in PFs than for FLFs but genes in both life cycle stages had low read numbers (Supplementary Table S. venezuelensis life cycle stages show that expression levels of SCP/TAPS-coding genes are higher at the life cycle stages inside the host i.e. the third stage larvae in the lungs (lL3), young parasitic females (yPF) and gravid parasitic females (gPF), compared with life cycle stages external to the host i.e. eggs, first/second stage larvae (L1/2) and free-living infective larvae stages (iL3) , SCP/TAPS, prolyl endopeptidase, transthyretin-like and trypsin inhibitors4 Fig. . The astStrongyloides species , 10 Biological Processes (BP) and 6 Cellular components (CC) GO terms were enriched amongst genes upregulated in the PF stage  activity is also enriched in the PF stage, supporting previous observations that cholinesterases have a putative role in Strongyloides parasitism4   upregulated in the PF stage of S. venezuelensis, across several further life cycle stages including eggs, larval stages, young parasitic adult (i.e. that do not carry eggs) and gravid parasitic adult stages of S. venezuelensis  PF, specifically at the mature, gravid stage or (ii) matured ovaries or eggs when inside, compared with outside, of the PF.Between one and six genes encoding Argonaute proteins were upregulated in the PF of all four –5) Fig. . In C. eStrongyloides nematodes possess families of Argonautes similar to those of the C. elegans families, including ERGO-1, ALG-3/4 and ALG-1/2 (microRNA pathway)  are absent for nematodes outside of phylogenetic nematode clade V to which C. elegans belongs14, including Strongyloides species15  proteins, were upregulated in PF compared with FLF . In comparison, no genes encoding SPOP-like were differentially expressed between PF and FLF for S. ratti and S. stercoralis. This suggests that the SPOP-like protein family has a role in parasitism or another feature associated with PF biology for the S. venezuelensis-S. papillosus subclade, but not the S. ratti-S. stercoralis subclade of Strongyloides nematodes. The SPOP-like family is greatly expanded in S. venezuelensis (48 genes) and S. papillosus (61 genes), compared to other nematodes including other Strongyloides and Parastrongyloides species, which possess between 0–2 SPOP-like encoding genes in their genomes3. The SPOP proteins are components of the cullin-RING-based BCR (BTB-CUL3-RBX1) E3 ubiquitin-protein ligase complex, commonly involved in proteasomal degradation of targeted proteins in the ubiquitin proteasome pathway (UPP). Interestingly, a greater number of genes encoding F-box proteins, which play a role in recruiting substrates to a core ubiquitin, are also upregulated in the PF of S. venezuelensis (10 genes) and S. papillosus (13 genes), than those upregulated in the PF of S. ratti (two genes) and S stercoralis (four genes). Together these results suggest that ubiquitination is upregulated in the PF of S. venezuelensis and S. papillosus, compared with S. ratti and S. stercoralis.In FLF Fig. . In S. pS. venezuelensis, S. papillosus, S. ratti and S. stercoralis show that the expanded SPOP-like gene family in S. venezuelensis and S. papillosus is distinct from the SPOP-like genes found in other nematodes , a conserved protein-protein interaction domain16, and a MATH/TRAF domain (IPR002083), involved in substrate recognition and binding17 - except for the four S. venezuelensis and S. papillosus genes in this cluster which encoded either one or neither of these domains. Genes in this conserved cluster were expressed, but not differentially so, between the PF and FLF stages. In total, 23 S. venezuelensis and 38 S. papillosus SPOP-like encoding genes coded for both a BTB/POZ and a MATH/TRAF domain, including 17 and nine genes, respectively, that were upregulated in the PF. Twenty-nine and eleven of the 31 SPOP-like genes upregulated in the PF stage of S. venezuelensis and S. papillosus, respectively, formed a distinct phylogenetic cluster, supporting that the expansion of this cluster is important in S. venezuelensis parasitism . Most strikingly, genes encoding cytochrome P450 proteins were distinctly upregulated in the PF transcriptome of S. venezuelensis. Specifically, seven genes (of 13 predicted in the genome) predicted to encode cytochrome P450 family 4 subfamily V member 2 (4V2) proteins and 12 genes (of 34 genes in the genome) encoding cytochrome P450 18a1 subfamily of proteins were upregulated in the S. venezuelensis PF transcriptome, but were not upregulated in the PF transcriptomes of S. papillosus, S. stercoralis, and S. ratti . Cytochrome P450s are a superfamily of heme proteins. Enrichment of the Molecular Function GO terms for electron carrier activity (GO0009055), heme binding (GO:0020037) and iron ion binding (GO:005506) exclusively in the PF (cf. FLF) transcriptome of S. venezuelensis but not the PF of the three other Strongyloides species further support a unique role of cytochrome P450s in the S. venezuelensis PF stage  and experimental infection of mice under laboratory conditions. S. venezuelensis and S. ratti belong to different phylogenetic subclades and are likely to have evolved parasitism of rats independently18. Genes upregulated in the PF of S. venezuelensis and S. ratti, but not S. papillosus and S. stercoralis may therefore represent gene families that are specifically important for infecting rodent hosts e.g. genes coding for proteins with a role in avoiding or manipulating the rodent immune response. Interestingly, from our comparative analysis of the PF and FLF transcriptomes of all four Strongyloides species, we found a small number of gene families that were specifically upregulated in the Strongyloides rodent-parasites (Supplementary Table S. venezuelensis and S. ratti PF (cf. FLF), but not upregulated in the PF of S. papillosus and S. stercoralis, with each gene family comprising 1–3 upregulated genes per species. Based on previous analyses, most of these genes formed orthofamilies with genes from S. papillosus and S. stercoralis3 i.e. where orthologous genes were not differentially expressed between the PF and FLF stages. In S. venezuelensis and S. ratti, the fold change difference in expression levels between PF and FLF stages, although significant, was relatively low . It is therefore possible that the gene families commonly upregulated in S. venezuelensis and S. ratti  may represent sampling differences rather than true features of the PF transcriptome associated with Strongyloides rodent parasitism. To include gene families that may not be recognised or have a predicted function, we compared the orthofamilies  that were uniquely upregulated in the PF (cf. FLF) in pairwise species combinations. The number of orthofamilies uniquely upregulated in one species compared to the three other species ranged between 165–459 in PF and 146–529 in FLF  which ranged between 9 and 39 orthofamilies. This suggests that the S. venezulensis and S. ratti PF transcriptomes are more similar to one another, outside of evolutionary subclades, and this may represent their ability to parasitise a common rodent host.Within the Strongyloides species has identified key gene families with a putative role in parasitism by Strongyloides species, including gene families likely to have a role in parasitism at the genus (e.g. WAGO-like), subclade (e.g. SPOP-like), and species (e.g. cytochrome p450) level. In particular, this study has identified WAGO-like Argonautes associated with a parasitic (compared to free-living) life cycle stage. Studies on the small RNAs pathways associated with a role in nematode parasitism have predominantly focused on the microRNA pathway19, which is not mediated by WAGOs. The WAGO-like coding genes upregulated in Strongyloides PF may therefore represent another pathway involved in parasitism, for example, involving the secretion of WAGO-mediated small RNAs into the host to manipulate host genes expression. Alternatively, this expression pattern could be due to an upregulation of WAGO-like Argonautes in the matured ovaries or eggs of gravid parasitic, compared with eggs that have been released and the matured ovaries and eggs inside free-living adult females. Little is known about differences that exist between the eggs inside parasitic versus free-living females. In the Strongyloides life cycle, PF reproduce by mitotic parthenogenesis and FLF reproduce sexually20 and the differential expression of the WAGO-like Argonaute may therefore be associated with a role in parthenogenic reproduction.Comparative analysis of the transcriptomes of four Strongyloides species presented here enables inferences to be made about the divergent evolution of parasitism of two distinct phylogenetic Strongyloides subclades . Upregulation of genes in the PF that are related to ubiquitation (i.e. SPOP-like and F-box-coding genes), coupled with an expansion of the SPOP-like gene family specifically in S. venezuelensis and S. papillosus suggests a putative role of the Ubiquitation Proteasome Pathway (UPP) in parasitism or features associated with the parasitic life cycle stage in this subclade. However, because the UPP is central to the regulation of many diverse cellular processes21, the exact role, if any, of UPP in parasitism or features associated with the parasitic life cycle stage of the S. venezuelensis-S. papillosus subclade is not clear and remains to be investigated.The comparison of transcriptomic data for four Strongyloides species share many similarities across their life cycle, for example, they all have a parasitic stage inhabiting the mucosa of the small intestine of their host and a free-living stage outside of the host. All known Strongyloides species parasitise tetrapod  hosts, but the hosts vary between Strongyloides species. We have identified genes coding for protein families that are uniquely upregulated in the PF of each species, which may represent adaptation to parasitising distinctly different hosts. It should also be noted that data for S. venezuelensis and S. ratti used in this analysis are from PFs raised in their natural rodent host, compared to S. papillosus and S. stercoralis PFs which were raised in permissive hosts . It is therefore possible that genes upregulated in the PF of S. papillosus and S. stercoralis may also represent a host-parasite interaction that has not co-evolved outside of laboratory conditions. Although S. venezuelensis and S. ratti both parasitise rodent hosts we found few gene families that are specifically upregulated in the PF of these two species, but not S. papillosus and S. stercoralis. Together, this suggests that S. venezuelensis and S. ratti have either evolved different molecular strategies to parasitise their rodent hosts, (represented by gene families that are uniquely upregulated in their respective PF transcriptomes) or they use similar genes and the proteins to infect rodents, but these are regulated at the post-transcriptional or post-translational level. Together, our results identify key gene families with a putative role in Strongyloides parasitism or features of the parasitic life cycle stage, and deepen our understanding of parasitism evolution among Strongyloides species.S. venezuelensis HH1 is used in this study, which have been maintained in the Parasitology laboratory at the University of Miyazaki. In brief, S. venezuelensis have been maintained in male Wistar rats by serial infection using subcutaneous injection of infective larvae prepared by faecal culture using filter paper9. Parasitic females were isolated from rat small intestine at 8 days post-infection (d.p.i). Faeces (approx. 1 g) collected from infected rats at 12 d.p.i were cultured on a 2% (w/v) agar plate at 25 °C for 3 days and FLF nematodes were collected. Twenty PF or 8–16 FLF were transferred to individual tubes containing 150 µL TRI reagent (Life Technology) and total RNA was extracted according to the standard procedures (Life Technology). RNA was then amplified using the SMARter Ultra Low amplification kit (Clontech) and sequencing libraries were constructed using the TruSeq Sample Prep kit according to the manufacturer’s recommended protocols (Illumina). The libraries were sequenced for 151-bp paired-ends on an Illumina MiSeq sequencer with MiSeq Reagent Kit v2 (300 cycles) using the standard protocol (Illumina) to obtain >15 million pair-end reads for each sample. RNAseq experiments were conducted in triplicate . Reads have been submitted to the DNA Data Bank of Japan (DDBJ) under BioSample accession numbers SAMD00096905-SAMD00096910.All the animal experiments were performed in accordance with the procedures approved by the Animal Experiment Committee of the University of Miyazaki under an approval no. 2009-506-6.S. venezuelensis RNAseq reads (this study) and S. papillosus data (downloaded from the European Nucleotide archive (accession no. PRJEB14543)) were aligned to the genome references  using TopHat222 using the same parameters as Hunt et al.3 for S. stercoralis and S. ratti (parameters: –a 6 –i 10 –I 20000–microexon_search–min-segment-intron 10–max-segment-intron 20000). Read counts per gene and fragments Per Kilobase of transcript per Million mapped reads (FPKM) were calculated using HT-seq.23 and cufflinks24 packages, respectively. Differential expression of genes was determined by edgeR25 analysis using R version 3.3.3. Common dispersion values 0.07379822, 0.1692273, 0.006412771 and 0.007270067 for S. venezuelensis, S. papillosus, S. ratti and S. stercoralis, respectively, were estimated for all genes using the edgeR package25 n.b. S. papillosus has a higher dispersal rate compared to the three other Strongloides species . The same methods and parameters for transcriptome analysis were used for all four Strongyloides species. RNAseq data of S. venezuelensis other stages were retrieved from DDBJ under BioProject accession number PRJDB3457.S. venezuelensis and S. papillosus PF and FLF life cycle stages were established using the TopGO27 package in R version 3.3.3. For comparative analyses with S. ratti and S. stercoralis, GO term enrichment analysis from Hunt et al.3 were used, which were analysed using the same method as described here for S. venezuelensis and S. papillosus.Enriched GO terms for Speckle-type POZ-like (SPOP-like) genes: the SPOP-like gene family was identified based on the predicted protein function as described by Hunt et al.3. Further analysis of protein domain predictions was carried out using InterProScan searches for BTB/POZ InterProScan domain (IPR000210) and MATH/ TRAF domain (IPR000210).Argonaute genes: Genes predicted to encode Argonaute proteins were identified by the presence of a PIWI domain (IPR003165) characteristic of Argonaute proteins and prediction of either a PAZ (IPR003100) or ribonuclease h-like domain (IPR012337).28 and phylogenetic trees were produced using RAxML with –m PROTGAMMAAUTO and 100 bootstraps29. For both phylogenies, the GAMMA ML model was selected as the best fit to the data. For the construction of phylogenetic trees, genes from 14 nematode species were used to represent species across multiple evolutionary clades. These species are S.venezuelensis, S. papillosus, S. stercoralis, S. ratti, P. trichosuri, Rhabditophanes sp., Caenorhabditis elegans, Necator americanus, Meloidogyne hapla, Trichinella spiralis, Ascaris suum, Brugia malayi, Bursaphelenchus xylophilus and Trichuris muris.Amino acid sequences of SPOP-like and Argonautes were individually aligned using MUSCLEStrongyloides species used in this analysis, and UniProt’s naming guidelines were used to assign protein names (http://www.uniprot.org/docs/nameprot), as described by Hunt et al.3.Families of genes grouped according to the predicted function of the protein they code for. The same parameters were used for the annotation of protein function for the predicted genes for each of the four 3 using EnsemblCompara30.Families of orthologous genes based on previous analysisSupplementary Fig. S1 to S5Supplementary Tables S1 to S14


AbstractArgyrellarichardsiae Veranso-Libalah & G.Kadereit, sp. nov. , is described and illustrated. Although the widespread Argyrellacanescens also occurs in Tanzania and northeastern Angola, A.richardsiae is morphologically most similar to Argyrellabambutorum known only from the Northwest of Cameroon, but differs by its indumentum of glandular trichomes on the entire plant (versus a mixture of stellate and glandular trichomes in other species of Argyrella), leaf-blades with serrulate margins (versus entire margins in A.bambutorum) and lateral nerves that become faint mid-way and never reach the leaf apex . A preliminary conservation status of Endangered (EN) is proposed for A.richardsiae following the IUCN Red List Categories and Criteria. A taxonomic key and distribution map of all Argyrella species is also included.A new species from the wet miombo woodlands of Tanzania and Angola,  Argyrella Naudin was first described by Dissotissect.Argyrella (Naudin) Triana. Later, D.canescens (E.Mey. ex Graham) Hook.f. as well as D.angolensis Cogn. to Dissotissubgen.Argyrella (Naudin) A.Fern. & R.Fern. These two species together with four other Dissotis species previously treated in “séries des Dissotis canescents” by HeterotisBenth.sect.Argyrella (Naudin) Jacq.-Fél. by Heterotissect.Argyrella . Although the phylogenetic relationships between these species were not well-resolved, all four species formed a well-supported clade in the Bayesian inference, maximum likelihood and parsimony analyses. In addition, their study of herbarium material morphologically supported the resurrection and updated circumscription of Argyrella with six species including five new combinations. Argyrella together with Guyonia Naudin, Melastomastrum Naudin, Anaheterotis Veranso-Libalah & G.Kadereit and Tristemma Juss. belong to the ‘Pseudoheterotis’ clade which consists mainly of herbs with persistent calyx lobes lacking intersepalar appendages. Argyrella is closely related to the monospecific Anaheterotis but is distinguished by having stellate and/or glandular trichomes on the entire plant (glabrous in Anaheterotis), entire to serrulate leaf margins (versus densely serrate margins ending in prominent ciliate trichomes in Anaheterotis). Also, Argyrella can be distinguished from Heterotis by its erect growth (versus a decumbent habit in Heterotis), calyx-tubes with an indumentum of stellate and/or glandular trichomes [versus stalked stellate emergences in Heterotis (except in H.decumbens (P.Beauv.) Jacq.-Fél. which has simple trichomes)], and paniculate inflorescences (versus flowers solitary or in cymes in Heterotis). The chromosome number n = 17 in A.canescens (E.Mey. ex Graham) Harv. and A.amplexicaulis (Jacq.-Fél.) Veranso-Libalah & G.Kadereit is the same as those counted in species of Melastomastrum and Tristemma , P , LISC (http://actd.iict.pt/) and COI , we have identified a wet miombo woodland species of Argyrella from Tanzania and Angola that is new to science, described and illustrated herein. All measurements were taken from dried specimens. The extent of occurrence (EOO) and area of occupancy (AOO) were calculated using GeoCAT (Argyrella.From our comparative study of herbarium collections through visits (BR and BRLU), loans , and online repositories: BM  , S. Bidgood, D. Sitoni, K. Vollesen & C. Whitehouse 3935 .TANZANIA. Katavi region: Mpanda district, 19 km on Mpanda-Uvinza road, seepage areas in tall Argyrella by having only glandular trichomes throughout the whole plant (versus a mixture of stellate and glandular trichomes in the other species), secondary branches arising at each node and leaves generally pointing downwards. It resembles A.bambutorum (Gilg & Ledermann ex Engl.) Veranso-Libalah & G.Kadereit but differs by having leaf-blades with serrulate margins (versus entire margins in A.bambutorum) and the primary pair of lateral nerves disappearing half-way between the base and the apex . Argyrellarichardsiae also differs from the widely distributed A.canescens and the Angolan endemic A.angolensis (Cogn.) Veranso-Libalah & G.Kadereit by having many new branches or buds arising at each node (versus unbranched herb in A.angolensis and A.canescens) and leaves sessile with amplexicaul bases generally pointing downwards  and only glandular trichomes on the hypanthium (versus dense stellate and glandular trichomes on the hypanthium of A.angolensis and A.canescens).The new species differs from all other species of PageBreakPageBreakStyle 22–25 mm long, glabrous. Stigma punctate. Fruit a capsule, dehiscent, ca. 6 mm long. Seeds cochleate, ca. 0.6 mm in diameter, numerous . TANZANIA. Mpanda district: Uruwira-Tabora road, Brachystegia woodland, Kambisama river, 1400 m, 30 September 1970 (fl & fr), Mrs H.M. Richards & S. Arasululu 26190 ; Mlele beekeeping reserve, riverine woodland, Iloba river, 6°47'56"S, 31°37'33"E, 1562 m, 7 May 2004, N.A. Mwangulango 1193 .ANGOLA. Huambo province: Longonjo, Lépi, Caála 1700m, 3 August 1940, (fr), Argyrellarichardsiae, she made a collection which is cited above in ‘Additional specimens examined’.The species epithet is in honour of Mary Alice Eleanor Richards , who collected extensively in Africa from 1951 to 1974 (Polhill and Polhill 2015). Of our new species Brachystegia Benth., Julbernardia Pellegr. and Isoberlinia Craib & Stapf trees of the subfamily Caesalpinioideae, Leguminosae. The wet miombo is found in areas of more than 1000 mm annual rainfall with an elevation of 1000–2500 m. A predominant wet miombo woodland vegetation is composed of riverine woodland along watercourses and marshes in poorly drained and/or low-lying areas, mainly characterised by alluvial soils , northern Zambia and southern Malawi and not just disjunctly between Angola and Tanzania. Argyrellarichardsiae like the other Argyrella species grows in marshes.This species is evidently endemic to the wet miombo woodlands of Mpanda district, southwestern Tanzania and Huambo province, central Angola Fig. . Miombo al soils . Also, wal soils . It is lEN): B2ab  EOO ~79 km2, AOO 32 km2. This species is only known from four collections in seepage, marshes or riverine woodlands (wet miombo woodlands) of the Mpanda district, Tanzania and Huambo province of Angola. Although cited on the collection label Mwangulango 1193 as a locally frequent herb in the Mpanda district, it is still a poorly collected species. As earlier suggested, A.richardsiae may also occur in wet miombo woodlands of southern DRC, northern Zambia and southern Malawi, but at the moment we think it is better to treat it as endangered until we are certain that this species is found in other places. Moreover, this species is only known from one collection in Angola since 1940. Generally, miombo woodlands are an important source of livelihood because they provide social, economic, and environmental beneﬁts PageBreaksuch as ﬁrewood, timber, medicinal plants, food, and catchment protection, among others. According to Proposed IUCN Red List Category: Endangered (KX889285 (ITS), KY248410 (psbK-psbL), KY284711 (accD-psaI) (see saI) see .Argyrellarichardsiae is similar to A.bambutorum but differs by having serrulate leaf margins, intersepalar appendages absent, and stamens dimorphic . The new species also differs from A.amplexicaulis, A.sessilis (Hutch. ex Brenan & Keay) Veranso-Libalah & G.Kadereit and A.angolensis by its many new branches or buds arising at each node . Argyrellarichardsiae has broadly ovate leaves versus lanceolate to linear in Argyrellacanescens and A.linearis (Jacq.-Fél.) Veranso-Libalah & G.Kadereit. Also, A.richardsiae has only glandular trichomes on the entire plant and leaves generally pointing downwards versus a mixture of stellate and glandular trichomes with leaves pointing upwards in the other species.


We are writing in response to the Correspondence from Tegtmeyer and Backert regarding the article by Helicobacter pylori-infected cells  and vacuolating cytotoxin (VacA), are required for pathological actions in ed cells . After ded cells . Previoued cells . Receptoed cells .H. pylori, but not in AZ-521 cells. We agree with their observations; we also observed that pCagA could not be detected in AZ-521 cells by standard western blotting using anti-pY antibody during infection with H. pylori (H. pylori infection could be detected in AZ-521 cells. When we carried out the infection assay in AZ-521 cells by challenging with H. pylori strains under several experimental conditions, we could detect pCagA by infection with wild-type H. pylori. On the other hand, this signal could not be detected during infection with a cagPAI-deleted mutant of H. pylori, indicating that this signal originated with pCagA or total CagA protein (fig. 4A in H. pylori was verified by two additional methods (fig. 4A and B in H. pylori-infected host cells, suggesting that the amount of precipitated SHP2 phosphatase correlated with the amount of pCagA (Tegtmeyer and Backert showed in their . pylori A. To addof pCagA . Second,of pCagA . Thus, tof pCagA B. When wof pCagA C. TherefH. pylori infection is not known. Therefore, it is not clear whether the concentrations of VacA used in our study correspond to the relative concentration on the surface of H. pylori-infected host cells. Although VacA concentrations that affect its biological functions in host cells, and physiological concentration during H. pylori infection, should be examined in further studies, we propose that VacA does not have a prolonged effect on induction of Src phosphorylation in AZ-521 cells (fig. 1A in The physiological concentration of VacA during H. pylori-infected host cells. Although there appears to be a problem regarding CagA phosphorylation, we used AZ-521 cells because this cell line is highly sensitive to VacA-induced actions and expresses several VacA receptors including RPTPα (H. pylori infection using these cell lines. Primary cell cultures and polarized cells with 3D structure, such as organoids generated from the biopsy of human gastric epithelium or animal experimental models, might be applicable for studies regarding the biological functions of virulence factors such as CagA and VacA in H. pylori-infected host cells.We also think that AGS cells represent a good model gastric epithelial cell line to study the biological functions of CagA in ng RPTPα . But, as