Rnaset2-based syngeneic in vivo assay. BALB/c mice were injected with parental, empty vector-transfected or murine Rnaset2-overexpressing mouse C51 or TS/A syngeneic cells and tumor growth pattern and immune cells distribution in tumor mass were investigated. Compared to control cells, mouse Rnaset2-expressing C51 cells showed strong delayed tumor growth. CD86+ M1 macrophages were massively recruited in Rnaset2-expressing C51-derived tumors, with concomitant inhibition of MDSCs and CD206+ M2 macrophages recruitment. At later times, a relevant expansion of intra-tumor CD8+ T cells was also observed. After re-challenge with C51 parental cells, most mice previously injected with Rnaset2-expressing C51 cells still rejected C51 tumor cells, suggesting a Rnaset2-mediated T cell adaptive immune memory response. These results point at T2 RNases as evolutionary conserved oncosuppressors endowed with the ability to inhibit cancer growth in vivo through rebalance of intra-tumor M1/M2 macrophage ratio and concomitant recruitment of adaptive anti-tumor CD8+ T cells.Human RNASET2 acts as a powerful oncosuppressor protein in in vivo xenograft-based murine models of human cancer. Secretion of RNASET2 in the tumor microenvironment seems involved in tumor suppression, following recruitment of M1-polarized macrophages. Here, we report a murine  RNASET2 gene encodes for an evolutionarily conserved, pleiotropic extracellular ribonuclease, whose secretion by cancer cells in the tumor microenvironment (TME) is likely involved in tumor suppression. Indeed, two independent in vivo xenograft-based murine models of human ovarian cancer have been previously used to demonstrate a marked RNASET2-mediated in vivo tumor suppression [RNASET2 affects several important cancer-related parameters  in a cell-autonomous manner, [RNASET2-mediated noncell-autonomous oncosuppressive role [RNASET2 overexpression in two independent human ovarian cancer cell lines, followed by challenging of immunodeficient mouse models with these cells, showed a marked tumor suppressive effect coupled with a RNASET2-mediated recruitment of M1-polarized host macrophages within the tumor mass [Hirudo verbana [The human pression ,2. It ha manner, ,4,5,6. Mive role . In factmor mass ,9, stron verbana , thus po+ T cells in cancer immunoediting and tumors evading via an adaptive immune resistance phenotype [The concept that the immune system can recognize and control tumor growth was mainly based on the many in vivo data related to immunoediting phenomenon in preclinical models and humans ,12, as whenotype . In the henotype ,18,19,20Macrophages represent key innate immune effectors fighting against pathogens and tumors, but they also have a role in the regulation of tissue homeostasis, repair and tumor progression . These cRnaset2 gene by overexpressing it in either mammary adenocarcinoma-derived TS/A or C51 colon carcinoma murine cells and injecting them subcutaneously in syngeneic BALB/c mice, using both empty vector-transfected (E) and parental (P) cells as a control. In light of the previously mentioned role of human RNASET2 in modulating the macrophage activation/polarization state, to further investigate the oncosuppressive role of T2 Ribonucleases in the context of a completely immunocompetent experimental model we report here the in vivo role of the murine Rnaset2-expressing C51 cells in comparison to control tumor cells-injected mice. Furthermore, four out of ten mice were able to completely reject primary full length Rnaset2-expressing C51 cells. Intriguingly, three out of four of these tumor-suppressed mice remained tumor-free when re-challenged with parental untransfected C51 cells, indicating the establishment in these mice of immune memory responses and thus, induction of adaptive anti-tumor T immune cells as well. Furthermore, a trend for an in vivo Rnaset2-mediated tumor suppressive effect was observed in TS/A cells as well.In this work, we report for the first time in a syngeneic mouse model a significant inhibition of tumor growth in mice injected with murine Rnaset2-mediated tumor suppression, we thoroughly investigated the tumor cell immune infiltrates in the C51 tumor model. Compared to control mice, M1 macrophages were massively recruited in the Rnaset2-expressing tumors at 2 weeks post-tumor inoculation, with a concomitant inhibition of both M2 macrophages and MDSCs influx, whereas at 3 weeks post-injection a statistically significant expansion of intra-tumor CD8+ T cells population in mice injected with Rnaset2-overexpressing C51 cells was observed. To ascertain the role of immune cells in + T helper (TH) type 1 cells and F4/80+ macrophages were observed when compared to both P and E C51-injected control mice. Moreover, a trend for expansion of spleen myeloid CD11b+ TNFα -producing cells was also detected.Moreover, in C51 tumor-rejecting mice a statistically significant expansion of spleen IFNγ-secreting CD4+ T cells.Taken together, these results support the role of T2 Ribonucleases as evolutionary conserved tumor suppressor genes endowed with the ability to inhibit cancer growth in vivo also in immunocompetent experimental models, through rebalance of intra-tumor M1/M2 macrophage polarization towards TNFα-producing M1-type macrophages and recruitment of adaptive CD8Rnaset2. As shown in Rnaset2 cDNA overexpression (TS/A or C51 “FL Rnaset2” clones). After a considerable screening, a few Rnaset2-overexpressing clones were obtained for both cell lines  and C51 colon carcinoma (C51 P) cells were first investigated by western blot analysis to assess their endogenous expression level of ll lines A,B and sRnaset2-overexpressing TS/A clones did not show a significant difference in their in vitro proliferation rate when compared to control, empty vector-transfected clones. However, when the same clones were challenged in vivo by subcutaneous injection in syngeneic BALB/c mice, a trend for Rnaset2-mediated decrease of tumor growth rate was observed  were inoculated in parallel with parental, untransfected C51 P and empty vector (C51 E) cancer cells in distinct groups of BALB/c mice in order to investigate after two months its in vivo capacity to resist a homologous re-challenge with C51 P cells and the features of infiltrating immune cells.Next, C51 mouse colon carcinoma cells overexpressing the murine Rnaset2 C51 cells were able to completely reject tumor cells and remained tumor-free for about two months  and cancer cells themselves ,28.Given their widely acknowledged functional plasticity, macrophages within the TME can carry out either pro-tumor or anti-tumor roles, as epitomized by the well-established M1 vs M2 polarization paradigm ,23,24,25RNASET2 gene as a potential player for in vivo M1/M2 imbalance regulation. Indeed, two independent xenograft-based experimental models of human ovarian carcinoma clearly showed the occurrence of a marked RNASET2-mediated in vivo tumor suppressive role, which was apparently based on the ability of cancer cells-derived RNASET2 protein to recruit M1 macrophages within the TME [Recently, we have defined the human  the TME . Moreove the TME . Therefo the TME .Despite being a key player in the control of cancer development and progression, the innate immune system has to establish a tight crosstalk with the adaptive immune system in order to carry out a fully effective role in either directions . It is therefore tempting to speculate that signaling molecules originally defined by their effects on the innate immune system (such as RNASET2) might actually be involved in triggering an adaptive immune response as well.Rnaset2 gene in syngeneic murine models. To this aim, two different mouse cell lines (breast adenocarcinoma-derived TS/A and colon carcinoma-derived C51) were chosen. Since our previous results indicated that human RNASET2 can carry out its oncosuppressive role to a different extent depending on the adopted experimental model [Rnaset2 by means of both in vitro and in vivo assays. To address this topic, we report here for the first time the results from in vivo experiments to decipher the role of the mouse Rnaset2, E vector, and P untransfected cancer cells.The in vitro cell proliferation rate and in vivo tumor growth rate in syngeneic BALB/c mice were thus evaluated for FL Rnaset2-mediated tumor suppression (which was observed by in vivo assays only), the less tumorigenic C51 cell line turned out to undergo a marked Rnaset2-mediated tumor suppression in both in vitro and in vivo experimental settings, and was therefore chosen for further investigation. The observed cell line-dependent effects of murine Rnaset2 is totally in keeping with our previous results with xenograft-based in vitro and in vivo assays using human RNASET2 [Taken together, the experimental data allowed us to conclude that, whereas the highly aggressive TS/A cell line was only partially sensitive to Moreover, since our previous investigations on human RNASET2 protein unveiled its ability to modulate in vivo the recruitment of myeloid cells in the TME (in particular by triggering M1-polarized macrophages recruitment in in vivo xenograft-based murine models of human cancer) [Rnaset2-overexpressing clones compared to control clones was observed, with several time points showing significant difference between FL Rnaset2 and control C51 E and P cells. C51 P and C51 E control clones also showed a slight difference in in vivo tumor growth rates as well, although statistically not significant. Such difference might be related to either the different culture conditions in which these cells were grown in vitro before being injected in syngeneic mice  or, alternatively, to empty vector-induced genome integration effects.In the C51 tumor model, a clear trend for a marked inhibition of tumor growth in mouse FL Rnaset2-expressing cells in terms of several immunological-related parameters, which in some cases reached statistical significance. We thus speculate that the significant differences between C51 P and C51 E cells emerged in most single immunological assays carried out by cytometry are in keeping with the non-significant trend emerged in in vivo for the much complex process of growth rate assay. It is moreover worth noting that the observed slight difference in the growth rate in vivo between C51 P and C51 E cells, showing a trend for a slightly more pronounced tumor growth in the former, was mirrored in most flow cytometry data shown in Rnaset2-mediated tumor suppression, also in light of the wide amount of literature data supporting such role for several members of the T2 RNase family.Thus, taken together we reckon that, notwithstanding a few discrepancies  of M1/M2 polarization ratio in C51 FL Rnaset2-injected mice, concomitant with inhibition of MDSCs, granulocytes and vessel numbers. The myeloid cell compartment appears to be a crucial determinant in the fate of tumor growth [We next analyzed, at two different time post-inoculation, immune cell infiltration of tumor masses of mice injected differently with C51 FL r growth , indeed r growth ,35,36. Cr growth . Moreover growth .+ TH1 cells, which are key activators in the priming phase, and CD8+ T cytolytic cells in the rejection effector response against tumor [Conversely, M1-like macrophage induction correlates with better control of tumor growth and also stimulation of anti-tumor protective adaptive T cells ,40, in pst tumor ,42,43,44Rnaset2-mediated rebalance of M1/M2 macrophages ratio, we conducted a flow cytometric assay on day 17 after tumor injection on tumor-infiltrating leukocytes in the three groups of C51 tumor model. We demonstrated that there was an increase of CD86+ M1/CD206+ M2 macrophage ratio in the TME of C51 FL Rnaset2 injected mice when compared to C51 E and P control tumors. This point is fundamental since reprogramming TAMs towards an M1 anti-tumor role actually represents one of most considered strategies for immune control cancer growth [To corroborate our results showing an in vivo r growth ,32,39,40IFNγ has a crucial role in the orchestration of an effective anti-tumor response, mainly by a direct role by decreasing proliferation and metabolic activity in tumor cells, as well as indirectly by up-regulating MHC class I and II molecules expression, inhibiting angiogenesis, and driving TH1 polarization by DCs ,46,47.Rnaset2-associated memory rejection correlates with systemic expansion of F4/80+ TNFα-producing M1 macrophages and triggering of CD4+ TH1 cells both in order of increment of their percentage and also for IFNγ-secreting cells. We therefore proceeded to characterize functional systemic immune responses in tumor-rejecting mice, in comparison to control tumor-bearing mice, studying in vitro activation of macrophages and T cells from spleens of differently groups of injected mice. Noteworthy, we found that Rnaset2 in a colon syngeneic mouse carcinoma model leads to a concerted anti-tumor immune response able to exert a tumor growth delay and rejection. Of note, this immune response appeared correlated with induction of innate cell compartment in the TME, in particular modification of M1/M2 balance ratio, decrease of number of MDSCs, tumor vessels inhibition and triggering adaptive immune CD8+ T effector cells.Taken together, these results indicate that overexpression of murine 3xFLAG_version2 Rev: 5′ AGAGGTTCTAGACTACTTGTCATCGTCATCCTTGTAATC 3′3xFLAG _version2 Fw: 5′ AACTGGCTCGAGGACTACAAAGACCATGACGGTGA 3′The pcDNA3 plasmid (Invitrogen) was chosen as backbone expression vector. The inducible vector was purchased from the VectorBuilder company  and usedE. coli strain. Plasmid DNA was purified and then sent to sequencing , before using it for the following steps. Different constructs, already available in our lab, bearing the murine Rnaset2 gene were used as templates for PCR reactions with the following primers:mRnaset2A Fw: 5′ TGCTAAGGATCCACCATGGCGCCG 3′mRnaset2A Rev: 5′ ACCTGAGAATTCATGTTGGGTCTTTGTAGGTGGA 3′mRnaset2A overlap Fw: 5′ AGCCGGGGGAGCAGCTGTCCTCCAGGCAGGAA 3′mRnaset2A overlap Rev: 5′ CTGCTTGGGGGACGGCTGCTCCCCTGGCTCA 3′The amplification product was digested with XhoI and XbaI, gel purified and cloned into the pcDNA3 vector before transforming in the DH5α E. coli strain. The amplification products were digested with EcoRI and BamHI, gel purified and cloned into the pcDNA3 vector (together with a synthetic oligo coding for a TEV protease recognition site with EcoRI and XhoI sticky ends) before transforming in the DH5α Rnaset2 gene (FL Rnaset2) followed by TEV and 3xFlag sequences. Plasmid DNA was purified and then sent to sequencing , before using it for the transfections. The resulting plasmid, together with its empty control counterpart (E), was used to stably transfect two BALB/c-derived murine cancer cell lines, TS/A (mammary adenocarcinoma) and C51 (colon origin).Collectively, the inserted coding region is represented by the full length murine 2 atmosphere. TS/A mammary adenocarcinoma and C51 mouse colon adenocarcinoma  cells , both of them of BALB/c origin, were cultured in DMEM with 10% FBS at 37°C, 5% CO5) cells were subcutaneously (s.c.) implanted in 8- to 10-week-old immunocompetent syngeneic BALB/c mice . Besides empty pcDNA3 vector-transfected cells, parental untransfected cells  have also been added to the experimental plan in in vivo cell transplantation assays, in order to include a further negative control assay. In particular, we wanted to exclude unpredictable in vivo side-effects due to random integration in the transfected cell’s genome of the empty vector. The tumor volume was determined using the formula: (d) 2 × D × 0.52, where d and D, are the short and long dimension (cm) of the tumor, respectively, measured with a caliper. Mice were bred and kept under standard conditions at the animal facility of the IRCCS Ospedale Policlinico San Martino. Housing, treatment, and killing of animals followed national legislative provisions for the protection of animals used for scientific purposes and an ethics committee approved the protocol (#299/2018-PR). Euthanasia was performed when the tumor reached a volume of about 1.5 cm3 by cervical dislocation after CO2 treatment.Stably-transfected clones were cultured in the basic medium supplemented with G418 (300 μg/mL for TS/A and 400 μg/mL for C51). TS/A and C51  was used to determine viable cell number based on the cellular conversion of a tetrazolium salt into a formazan product. The absorbance of the solubilized formazan product  was recorded using a 96-well plate reader daily over a 7- or 10-day period. Samples were seeded in triplicates.The Cell TiterAdherent cells were mechanically scraped in PBS in the presence of 5 mM EDTA and resuspended in lysis buffer  supplemented with protease inhibitors cocktail . Quantification of total proteins was performed with Bradford reagent , using bovine serum albumin as standard. For the SDS-PAGE analysis, 70 μg of intracellular lysate were loaded. Immunoblot analysis was performed using standard procedures and detected with a chemiluminescent substrate, WESTAR ETA C ULTRA 2.0, Cyanagen . Antibodies used were: Rabbit monoclonal anti-FLAG (Sigma Aldrich); HRP anti-rabbit IgG .Spleen cells were isolated by scissor dissociation, collected in RPMI 1640–10% FBS, and filtered using a 70 μm cell strainer (Becton Dickinson (BD), San Diego, CA, USA). Erythrocytes in the splenic preparations were removed by hypotonic lysis with NH4Cl–KHCO3–EDTA solution. Tumor infiltrating leucocytes from C51 colon carcinoma were obtained by cutting the tumor mass into small pieces followed by incubation (1 g/10 mL) with a mixture of enzymes dissolved in serum free RPMI: collagenase type I (0.5 mg/mL), collagenase type IV (0.5 mg/mL), and hyaluronidase (0.25 mg/mL), all from Sigma Aldrich, and DNase I (0.1 mg/mL) from Roche  for 30 min at 37 °C. Undigested material was removed by filtration using a 70 μm cell strainer (BD) and the single cell suspensions were recovered and washed by centrifugation in RPMI 1640–10% FBS to remove residual enzymes.Spleen cells or tumor infiltrating leucocytes were first incubated with an FcR blocking reagent  for 10 min. Afterward, cells were labeled with anti-CD4 (clone GK1.5), anti-CD8 (clone 53-6.7), and anti-CD11b (clone M1/70) mAbs, all from eBioscience ; anti-F4/80 (clone BM8) and anti-CD86 (clone GL-1) mAbs from BioLegend , and anti-CD206 (clone MR5D3) mAb from BIO-RAD. Briefly, after physical parameter setting (Forward and Side Scatter), and using anti-LCA CD45 (clone 30-F11) mAb from BioLegend mAb for tumor infiltrating leucocytes analysis, lymphocyte and myeloid populations were identified and then M1, M2, macrophages and T cells were evaluated by mAb-specific staining and assessed by FACS analysis using CELLQUEST or DIVA software (BD). The direct immunofluorescence was performed using FITC-, PE-, or allophycocyanin-conjugated specific mAbs. Negative controls included directly labeled FITC-, PE-, and APC-conjugated isotype-matched irrelevant mAbs (BD).6/mL in RPMI 1640–10% FBS, and stimulated with 10 ng/mL phorbol 12-myristate 13-acetate  and 500 ng/mL ionomycin (Sigma-Aldrich) in the presence of monensin (BD) during 4-h incubation at 37 °C, and then treated with Cytofix/Cytoperm fixation and permeabilization kit (BD). For macrophage stimulation, spleen cells were seeded at 106/mL in RPMI 1640–10% FBS, and stimulated with LPS 100 ng/mL (Sigma-Aldrich) in the presence of monensin during 4 h incubation at 37 °C. The permeabilized cells were intracellularly stained with the PE-conjugated cytokine-specific mAbs anti-IFNγ (clone XMG1.2), and anti-TNFα (clone MP6-XT22), all from BD; or isotype-matched irrelevant mAbs from BioLegend.Spleen cells from different groups of BALB/c mice were analyzed for cytokine content as previously described . Briefly2/field. The results are expressed as cells or vessels number per high magnification microscopic field . Images of slides were acquired using Aperio AT2 scan (Leica).Cryostat sections (6 μm thick) were air-dried and fixed in cold acetone for 10 min. Immunostaining was performed using a previously described procedure  and the p < 0.0001, *** p < 0.001, ** p < 0.01, and * p < 0.05 were considered significant. Data were analyzed for statistical significance using two-way ANOVA with Bonferroni’s posttest or nonparametric Mann–Whitney by Prism Graphpad software . All error bars represent SEM. Values of **** In this work, we provide the first in vivo evidence supporting the role of a member of the highly conserved T2 ribonuclease gene family in tumor suppression by means of the functional involvement of molecular and/or cellular effectors belonging to both the innate and adaptive immune systems. Rnaset2 gene and subsequently challenged in syngeneic BALB/c mice, we could confirm the marked tumor suppressive role of this gene , but we also unveiled that Rnaset2-mediated tumor suppression is associated not only with an increase in the intratumor M1/M2 macrophages ratio, but also with the establishment of a tumor suppressive immunological memory, which clearly entails adaptive immunity as a further biological weapon exploited by this highly conserved, pleiotropic gene to carry out its oncosuppressor activity.By developing a murine experimental model where C51 mouse colon carcinoma-derived cells were engineered to overexpress the mouse


MS188 and MS1 are essential anther tapetum transcription factors; MS188 directly regulates MS1, which controls the expression of pollen coat protein genes, which are key components of the pollen wall. MS1 expression driven by the MS188 promoter in ms188, indicating that MS1 is sufficient to activate their expression; however, additional MS1 downstream factors appear to be required for high-level sPCP expression. Our ChIP, in vivo transactivation assay, and EMSA data indicate that MS188 directly activates MS1. Together, these results reveal a regulatory cascade whereby outer pollen wall formation is regulated by MS188 followed by synthesis of sPCPs controlled by MS1.Sporophytic pollen coat proteins (sPCPs) derived from the anther tapetum are deposited into pollen wall cavities and function in pollen–stigma interactions, pollen hydration, and environmental protection. In Arabidopsis, 13 highly abundant proteins have been identified in pollen coat, including seven major glycine-rich proteins GRP14, 16, 17, 18, 19, 20, and GRP–oleosin; two caleosin-related family proteins (AT1G23240 and AT1G23250); three lipase proteins EXL4, EXL5 and EXL6, and ATA27/BGLU20. Here, we show that GRP14, 17, 18, 19, and EXL4 and EXL6 fused with green fluorescent protein (GFP) are translated in the tapetum and then accumulate in the anther locule following tapetum degeneration. The expression of these sPCPs is dependent on two essential tapetum transcription factors, MALE STERILE188 (MS188) and MALE STERILITY 1 (MS1). The majority of sPCP genes are up-regulated within 30 h after MS1 induction and could be restored by  Pollen from the triple mutant of these (pcp-bα/β/γ) displays a substantially reduced hydration rate on stigmas, delayed pollen tube growth, as well as weakened anchoring to the stigmatic surface  (Zea mays (maize)  , 2+-binding protein (AT1G23240), which is now known as a caleosin-related family protein, and two putative receptor kinases (AT3G21920 and AT4G20670), which are now annotated as cysteine-rich repeat secretory proteins (CRRSP18 and CRRSP41) , which locates to Cluster #7, has no homology with any of the adjacent lipases, but shows co-expression with EXL4–EXL6 . Several transcription factors that are expressed specifically in the tapetum have been functionally characterized  line showed that only low levels of sPCP expression could be obtained 30 h post-MS1 induction, suggesting that additional factors, that may be induced by, or at least are downstream of MS1, are needed for late full high-level expression of the sPCPs. This work has revealed the regulatory cascade controlling pollen coat formation and shown that these major sPCPs are synthesized in the tapetum under the control of MS1. It has also indicated that there is temporal regulation of their expression to enable precise control of the biosynthesis of wall materials, and that they are subsequently deposited onto the outer pollen wall regulated by MS188 for functional pollen formation.In this study, we examined the subcellular localization of six sPCPs  using translational green fluorescent protein (GFP) fusions and determined that all were initially synthesized in the tapetum, but were later observed to fill the whole locule and surround the microspores. Additionally, we studied the regulatory cascade controlling their synthesis erecta (Ler-0) were used as the wild-type control. Seeds were sown on vermiculite and germinated for 3 d at 4 °C. The plants were grown under 16 h light/8 h dark conditions in a 22 °C growth room. T-DNA insertion mutant lines were obtained from the ABRC and NASC. Transgenic plants were generated via Agrobacterium tumefaciens-mediated transformation (–1 hygromycin B.Arabidopsis accessions Columbia (Col-0) and Landsberg ormation  and selems1 complementation, the 3.476 kb MS1 genomic fragment (993 bp promoter and 2483 bp MS1 gene) was amplified using KOD DNA polymerase (Toyobo) with primer set MS1 com-F/MS1 com-R. The oligonucleotides used for complementation and other assays in this study are listed in JXB online. Primers were synthesized by Generay. After verification by sequencing (Genomics), the fragment was cloned into the pCAMBIA1300 binary vector (Cambia) and subsequently introduced into the heterozygous +/ms1 plants, as described previously  and GR is driven by a 993 bp upstream sequence of MS1 and followed by a NOS terminator in the inserted T-DNA.The ms188, and ms1 inflorescences using TRIzol (Life Technologies) following the manufacturer’s instructions. First-strand cDNA was synthesized from 1 μg of total RNA using poly(dT)12–18 primer, AMV transcriptase, and accompanying reagents (Takara) for 15 min at 42 °C. Quantitative reverse transcription-PCR (qRT-PCR) was performed using gene-specific primers  values.The RNA was extracted from Col-0, mers see  and SYBRms1-1 and MS1-GR plants  and Ler-0 wild type. The DEX treatment  was performed when 0 h inflorescences were sampled. Ten complete inflorescences were collected from a single plant at each time point to represent one single sample. The opened floral buds were removed from the inflorescences. Samples were then preserved in liquid nitrogen in a –80 °C freezer before RNA extraction. Total RNA was extracted from flower buds using the QIAGEN RNeasy Mini Kit  in accordance with the manufacturer’s instructions. Three biological replicates and two technical replicates were used to set up the qRT-PCRs.Whole inflorescences with opened flower buds removed were collected for RNA isolation from pMS188:MS188-GFP and pMS188:4×MYC-MS188 transgenic lines as described by –ΔCt was taken as the fold enrichment. Primers for ChIP-qPCR analysis are listed in The ChIP procedure was performed on flower buds of MS188 gene was amplified using the primer pair MS188PMAL-F/R cloned into the pMAL-c5× vector . The fusion protein was expressed and purified according to the manufacturer’s instructions. The DNA fragment containing the MYB-binding site in the MS1 promoter was amplified and a biotin-labelled and competitor probe, named probe MS1-1 and MS1-2, were produced. EMSA was performed with a LightShift Chemiluminescent EMSA Kit (Thermo Scientific) according to the manufacturer’s instructions. The relevant primer sequences are listed in To obtain the maltose-binding protein (MBP)–MS188 protein for the EMSA experiments, the full-length fragment of the MS1 sequence was first TA TOPO (Thermo Fisher Scientific) cloned into pCR8 and LR cloned into the effector vector. The MS188 coding sequence was Gateway cloned into the pUB-DEST vector  and BioTek Synergy LX Microplate Reader in accordance with the manufacturer’s instructions.For the dual-luciferase  assay, the pGreenII 0800 LUC plasmid  and the National Centre for Biotechnology Information with the following accession numbers: MS1 (AT5G22260), MS188/MYB80/MYB103 (AT5G56110), MYB99 (AT5G62320), EXL4 (AT1G75910), EXL5 (AT1G75920), EXL6 (AT1G75930), ATA27/ BGLU20 (AT1G75940), Caleosin (AT1G23240), CRRSP18 (AT3G21920), CRRSP41 (AT4G20670), GRP14 (AT5G07510), GRP16 (AT5G07540), GRP17 (AT5G07530), GRP18 (AT5G07520), GRP19 (AT5G07550), GRP20 (AT5G07560), and GRP-Oleosin (AT5G07600).Sequence data were archived in TAIR , followed by high accumulation in the locule at the time of tapetum PCD (stages 11–12)  . Howevers 11–12) , f. Signs 11–12) . In addis 11–12) . These dms1 mutants, a reduction of GRP expression occurs and pollen coat formation is not observed  have been identified as essential for tapetum development and pollen formation , Fig. 3a and ms1 . A re-an mutants . It is wray data , suggestMS1-GR line (ms1-1/pMS1:MS1-GR). MYB99 was used as a positive control for MS1 induction since it has been previously reported as a direct regulatory target of MS1  and CCAACC (–664 to –659) , containing the predicted MS188-binding sites (CCAACC and AAACCA), was enriched  but not Probe-2MS1 (CCAACC)  and the 35S-promoter driven Renilla luciferase (REN), and a helper vector expressing the p19 silencing suppressor, were co-transformed into N. benthamiana leaves. The MS188 transactivation level of Probe-1MS1 was measured by normalizing the signal of the experimental LUC reporter to that of the internal control REN. By using a constitutively expressed REN reporter as an internal reference reporter, the experimental variability caused by differences in cell transformation efficiency and viability was minimized  . In our enriched , c. On tobserved , c. A reobserved  and two observed . The res(CCAACC) . When th(CCAACC) . To furtinimized . Our Duacontrols . These rms188 and ms1 mutants, and induction of MS1 increased expression of many of the sPCPs , SLR1-BP1/2, PCP-A1(PCP7), and PCP-Bs, have also been identified. Arabidopsis pollen coat cysteine-rich proteins (CRPs), the PCP-Bs, have been reported to be involved in pollen hydration  for sporopollenin synthesis and sexine formation (During anther development, the tapetum acts in multiple roles in pollen formation, providing nutrition for microspore development, secreting hydrolases for tetrad callose wall dissolution, and supplying structural components such as sporopollenin and pollen coat for pollen walls . The genof MS188 . Therefoof MS188 . In thisMS1 as the final transcription factor in this pathway is involved in the control of several events such as tapetum PCD and tryphine synthesis in late pollen maturation . TherefoJXB online.Supplementary data are available at Fig. S1. sPCP genes that showed no significant up-regulation 30 h post-DEX induction in the MS1-GR line.pMS1:MS1-GFP complementation plants.Fig. S2. MS1 expression in Fig. S3. Expression and purification of MS188 proteins.Table S1. Primers used in this study.eraa219_suppl_Supplementary-Data-FilesClick here for additional data file.


Picea abies) embryonic callus and non-embryogenic callus using CUT&RUN, which is a powerful method for chromatin profiling. Here, we show that H3K27me3 mainly accumulated in genic regions in the Norway spruce genome, similarly to what is observed in other plant species. Interestingly, H3K27me3 levels in embryonic callus were much lower than those in the other examined tissues, but markedly increased upon embryo induction. These results show that H3K27me3 levels are associated with the embryogenic potential of a given tissue, and that the early phase of somatic embryogenesis is accompanied by changes in H3K27me3 levels. Thus, our study provides novel insights into the role of this epigenetic mark in spruce embryogenesis and reinforces the importance of PRC2 as a key regulator of cell fate determination across different plant species.Epigenetic reprogramming during germ cell formation is essential to gain pluripotency and thus embryogenic potential. The histone modification H3K27me3, which is catalysed by the Polycomb repressive complex 2 (PRC2), regulates important developmental processes in both plants and animals, and defects in PRC2 components cause pleiotropic developmental abnormalities. Nevertheless, the role of H3K27me3 in determining embryogenic potential in gymnosperms is still elusive. To address this, we generated H3K27me3 profiles of Norway spruce ( PRC2-mediated H3K27me3 is associated with embryogenic potential in Norway spruce, and the early phase of somatic embryogenesis is accompanied by changes in H3K27me3 levels. Picea abies) is a common conifer species that is ecologically and economically important in Europe. In Norway spruce, two different types of callus can be generated: embryogenic callus (EC), usually induced from immature embryos, and non-embryogenic callus (NEC), induced from vegetative seedling tissues  contains histone methyltransferase activity, and is responsible for the tri-methylation of lysine 27 of histone H3 (H3K27me3). This histone modification is a repressive epigenetic mark observed in developmental regulatory genes and environmental responsive genes  and ABSCISIC ACID INSENSITIVE 3 (ABI3) transcription factor genes display low H3K27me3 in EC compared with NEC. Interestingly, a 1-week period in embryo-inducing medium was sufficient to provoke alterations in H3K27me3 profiles of both EC and NEC. Notwithstanding, these two types of callus displayed large differences in H3K27me3 profiles in response to somatic embryo induction. Thus, we revealed that two different types of calli in Picea abies have distinct chromatin profiles, suggesting that the genome-wide H3K27me3 status in EC underlies its high pluripotency of cell differentiation.In both EC and NEC, the H3K27me3 mark was enriched in genic regions, rather than in intergenic regions or transposable elements. Nearly half of H3K27me3-enriched genes were common between EC and NEC; however, many genes showed lower H3K27me3 levels in EC before somatic embryo induction when compared with other samples. For example, some homologues of Picea abies L. Karst) embryogenic culture cell line (EC) was established from immature embryos by a previously described method  and a Leica camera DFC490.Saccharomyces cerevisiae genomic DNA) was added to each sample after extracting MNase-digested DNA. The read counts of spike-in DNA, which mapped to the S. cerevisiae reference genome, were well correlated with total read counts in each sample.Nuclei were isolated as described previously with the exception that the MEB buffer contained no EGTA  was carrP. abies whole-genome v1.0 (Congenie.org) and merged if they were mapped in the same contigs. (ii) The merged fragments with a size longer than 700 bp were discarded. (iii) The contigs of genome sequences were split by 500 bp bins. (iv) The read numbers overlapping each bin were counted and P-values were calculated using a binominal test by each bin. (v) Obtained P-values were adjusted by p.adjust packages in R using the Benjamini–Hochberg method. (vi) The bins with false discovery rate ≤10−25 were selected. Peaks were merged if their distances were shorted than 100 bp. The genes that overlapped with H3K27me3 peaks were defined as H3K27me3-enriched genes. The H3K27me3 signal intensity per gene was defined as reads per kilobase per billion mapped reads over gene regions, including 500 bp extensions in both upstream and downstream orientations. For genomic features, transposable element sequences were annotated as described before (http://congenie.org/). To calculate the Shannon entropy the following transcriptome datasets were used for organ specificity: ENA ID: ERS235801 , ERS235809 , ERS235803 (needles from 2009), ERS235807 , ERS235808 , ERS235811 (stem from vegetative shoots), ERS235802 (vegetative shoots), and ERS235821 , and for condition specificity: ENA ID: ERS235803 (needles from 2009), ERS235804 (needles from 2008), ERS235805 (infected needles), ERS235814 (needles from dried twig), ERS235815 (needles girdled twig), ERS235817 (early morning needles), ERS235818 (mid-day needles), ERS235819 (late afternoon needles), and ERS235820 (night needles)  The paired-end reads were separately mapped by BWA (v0.7.17)  to the Phttps://genomevolution.org/coge/), Phytozome (v1), and NCBI, were searched for homologues in Amborella trichopoda, Persea americana, Populus trichocarpa, Oryza sativa ssp. japonica, Zea mays, Picea abies, Picea glauca, Picea sitchensis, Pinus pinaster, Pinus sylvestris, Pseudotsuga menziesii, Taxus baccata, Cycas micholitzii, Ginkgo biloba, Gnetum montanum, Selaginella moellendorffii, Sphagnum fallax, Marchantia polymorpha, Physcomitrella patens, and Chlamydomonas reinhardtii  homologues in the plant kingdom, using tblastx (P. abies genes of interest was used for gene ontology (GO) analysis. The Arabidopsis GO annotation was downloaded from TAIR (https://www.arabidopsis.org). The hypergeometric P-values were calculated based on the frequency of each GO term in P. abies genes of interest with Arabidopsis homologues over the whole set of P. abies genes with Arabidopsis homologues. Enriched GO terms were summarized using REVIGO , we adapted the previously developed CUT&RUN method  to this Among the five tissue samples, a total of 10 511 genes had H3K27me3 peaks within the gene body or in adjacent regions . Each tiP. abies gene. Approximately 65% of the total P. abies gene models had homology to at least one Arabidopsis gene based on a BLAST search. We then grouped the Arabidopsis homologues based on the presence of H3K27me3. Nearly 40% of P. abies genes showing homology to an Arabidopsis H3K27me3-marked gene were also H3K27me3-marked (P. abies homologous genes of non-H3K27me3 targets in Arabidopsis (less than 15%). Approximately 10% of orphan genes, which are specific for the gymnosperm lineage, had H3K27me3 marks , CURLY LEAF (CLF), and MEDEA (MEA). The latter is expressed only during the reproductive phase and is responsible for parent-of-origin-specific gene expression in the endosperm , lgorithm . In bothlgorithm . Interesde novo H3K27me3 present in callus  between EC and NEC, we identified orthologues of DMGs in Arabidopsis and then analysed whether specific GO terms were enriched among these orthologous genes. Among the 789 genes that were H3K27me3-marked in non-induced EC, but not marked in non-induced NEC, GO terms related to response to metabolism, and in particular flavonoid metabolism, were most highly enriched . On the PLETHORA 4/BABY BOOM (PLT4/BBM) and PLETHORA 5/EMBRYO MAKER (PLT5/EMK), which are AP2-type transcription factors (AGAMOUS-LIKE 15 (AGL15) and the LEAFY COTYLEDON genes LEC1 and LEC2 are also known embryonic markers, and overexpression of these genes increases the embryogenic potential of Arabidopsis vegetative tissues (ABI3 (MA_66505g0020), DRNL , CRF4 (MA_10432800g0010), CUC2 (MA_16595g0010), and FUS3 (MA_68722g0010) accumulated higher levels of this histone mark in NEC. Interestingly, a second homologue of FUS3 (MA_823242g0010) and WIP5 (MA_849g0010), as well as one of the ANT homologues (MA_32193g0010) had higher H3K27me3 levels in EC  . Our valcis-elements associated with H3K27me3 deposition in Norway spruce are not similar to those described in Arabidopsis, AT-rich motifs and REF6 DNA-binding motifs were detected in our dataset. This co-localization of REF6 binding motif-like sequences and H3K27me3 suggests that, similarly to Arabidopsis, the H3K27me3 catalysis mechanism could be based on cis-regulatory sequences. Nevertheless, this remains to be formally tested: considering that the H3K27me3 distribution is usually broader than the PRC2 binding site itself (cis-regulatory elements for H3K27me3 target genes. Therefore, a high-quality genome assembly and direct binding assays using PRC2 components will provide a better resolution for the identification of cis-regulatory elements such as the PREs identified in Arabidopsis. A previous study of chromatin conformation in a relatively large genome revealed that H3K27me3 is associated with chromatin domain borders (We found that some H3K27me3 target genes are similar between Arabidopsis and Norway spruce, supporting the conservation of the epigenetic mechanisms regulating these genes. At the same time, gymnosperm-specific genes also show H3K27me3, suggesting that H3K27me3 is an important epigenetic player in the regulation of species-specific pathways . AlthougPrevious empirical evidence showed that non-induced EC and NEC have different embryogenic potentials. This study validates these observations at the molecular level and adds a possible functional explanation. Here we detected that non-induced EC and NEC had largely different genes being marked by H3K27me3 , and thaBrassica napus and Hordeum vulgare (In some angiosperm species, it has been reported that heterochromatic epigenetic marks are associated with SE initiation rate. For example, perturbations of DNA methylation or H3K9me2 by chemicals facilitate embryogenesis from microspores in Associations between peroxidase activity and embryogenic potential have been observed in both angiosperms and gymnosperms . Also geGenerally, somatic embryogenesis takes 6–8 weeks after somatic embryo induction. We showed that both EC and NEC altered their H3K27me3 profiles only 1 week after somatic embryo induction, and that NEC and EC showed a different H3K27me3 accumulation profile in response to somatic embryo induction. While the H3K27me3 profile of induced EC resembled that of non-induced EC, NEC showed drastic changes in H3K27me3 target genes after induction. Considering that NEC reduces its proliferation and turns brown 2–3 weeks after SE induction, these large changes in the H3K27me3 profile might reflect a senescence program of the cells . Indeed,In this study, we demonstrated that the genome-wide level of H3K27me3 in EC is extensively lower than in NEC and seedlings. Low H3K27me3 levels, together with the action of phytohormones such as 2,4-D might be the essential qualifications ensuring somatic embryogenesis potential in gymnosperms as well as in angiosperms (JXB online.The following supplementary data are available at Fig. S1. Morphology of EC and NEC 2 weeks after somatic embryo induction.Fig. S2. Percentage of genes with H3K27me3 modiﬁcation in each auxin-related gene family.Fig. S3. Phylogenetic analysis of putative H3K27me3 methyltransferases in gymnosperms.cis-element sequences associated with H3K27me3 peaks.Fig. S4. Identification of Fig. S5. Expression of representative SE-related genes is anti-correlated with their H3K27me3 level.Table S1. Sequencing data list.Table S2. List of primers used in this study.eraa365_suppl_Supplementary_MaterialClick here for additional data file.


In July 2018, a novel avian influenza virus (A/Mandarin duck/South Korea/KNU18-12/2018(H11N9)) was isolated from Mandarin ducks in South Korea. Phylogenetic and molecular analyses were conducted to characterize the genetic origins of the H11N9 strain. Phylogenetic analysis indicated that eight gene segments of strain H11N9 belonged to the Eurasian lineages. Analysis of nucleotide sequence similarity of both the hemagglutinin (HA) and neuraminidase (NA) genes revealed the highest homology with A/duck/Kagoshima/KU57/2014 (H11N9), showing 97.70% and 98.00% nucleotide identities, respectively. Additionally, internal genes showed homology higher than 98% compared to those of other isolates derived from duck and wild birds. Both the polymerase acidic (PA) and polymerase basic 1 (PB1) genes were close to the H5N3 strain isolated in China; whereas, other internal genes were closely related to that of avian influenza virus in Japan. A single basic amino acid at the HA cleavage site (PAIASR↓GLF), the lack of a five-amino acid deletion (residue 69–73) in the stalk region of the NA gene, and E627 in the polymerase basic 2 (PB2) gene indicated that the A/Mandarin duck/South Korea/KNU18-12/2018(H11N9) isolate was a typical low-pathogenicity avian influenza. In vitro viral replication of H11N9 showed a lower titer than H1N1 and higher than H9N2. In mice, H11N9 showed lower adaptation than H1N1. The novel A/Mandarin duck/South Korea/KNU18-12/2018(H11N9) isolate may have resulted from an unknown reassortment through the import of multiple wild birds in Japan and Korea in approximately 2016–2017, evolving to produce a different H11N9 compared to the previous H11N9 in Korea (2016). Further reassortment events of this virus occurred in PB1 and PA in China-derived strains. These results indicate that Japanese- and Chinese-derived avian influenza contributes to the genetic diversity of A/Mandarin duck/South Korea/KNU18-12/2018(H11N9) in Korea.  Influenza viruses are highly genetically diverse because of the accumulation of mutations, frequent reassortment among gene segments, host switching ability, and consistent antigenic drift or shift ,2. BasedUnlike various combinations such as H2N2, H3N2, and H1N1, which threaten human health, H11N9 strains have mostly unknown effects for either avians or humans. H11N9 strains were first isolated in Europe (Great Britain) in 1982 , after wBased on the Global Initiative on Sharing All Influenza Data (GISAID) data, few cases have been detected in South Korea as the influenza H11 subtype, which were H11N4 , H11N6, Some studies have described that H11N9 isolated in China can replicate not only in avian but also in mammalian cells , and eveTherefore, routine surveillance of H11N9 is required, although no human H7N9 strains have been found in Korea. Notably, the H11N6/KU/2/2001 strain exclusively infects swine in Korea. As influenza virus has regularly evolved to infect various hosts, we examined the genetic diversity of AIV gene pools. In this study, the full genome of an H11N9 strain isolated from the feces of Mandarin ducks during surveillance in July 2018 in Korea was analyzed and characterized. Fresh feces were collected from wild birds using sterile swabs in South Korea in July 2018. The collected samples were stored at 2–8 °C, and then shipped to the laboratory within 12 h for further analysis.The fecal samples were resuspended in phosphate-buffered saline (pH 7.4) supplemented with antibiotics (100 U/μL of penicillin and 100 mg/μL of streptomycin) and thoroughly mixed by vortexing, followed by centrifugation at 3000 rpm for 10 min at 4 °C. The supernatants were filtered through hydrophilic polyether sulfone membrane filters . The filtered aqueous solution was inoculated into the allantoic cavities of specific pathogen-free (SPF) 10-day-old embryonated chicken eggs , which were then incubated at 37 °C for 72 h under humidified conditions and chilled at 4 °C overnight. The allantoic fluids from the inoculated eggs were checked for the presence of influenza A virus by an HA assay as previously described. Viral RNA was extracted directly from allantoic fluid samples positive for HA activity using the RNeasy Mini Kit  according to the manufacturer’s instructions. The RNA was eluted in 60 μL of RNase-free water to which 20 units of RNase inhibitor was added for storage at −80 °C until use. As a negative control, sterile water was added rather than the specimen.Additionally, to evaluate influenza growth and determine the subtype, reverse transcription polymerase chain reaction (RT-PCR) for influenza A virus was performed by amplification of the matrix gene-coding sequence following World Health Organization guidelines . RT-PCR NGS was conducted by GnCBIO  following the Hiseq X method as previously reported . BrieflyFor cluster generation, the library was loaded into a flow cell in which fragments were captured on a lawn of surface-bound oligoes complementary to the library adapters. Each fragment was then amplified into distinct, clonal clusters through bridge amplification. When the cluster generation was complete, the templates were used for sequencing.Illumina SBS technology utilizes a proprietary reversible terminator-based method that detects single bases as they are incorporated into DNA template strands. As all 4 reversible, terminator-bound dNTPs are present during each sequencing cycle, natural competition minimizes incorporation bias and greatly reduces raw error rates compared to other technologies. The result is highly accurate base-by-base sequencing that virtually eliminates sequence context-specific errors, even within repetitive sequence regions and homopolymers.Sequencing data was converted into raw data for analysis.https://www.ncbi.nlm.nih.gov/genbank/) and GISAID (http://www.gisaid.org). Homology analysis of nucleic acids was performed on the NCBI and/or GISAID website with BLAST. The phylogenetic relationships of the aligned sequences for eight gene segments were determined by alignment using MUSCLE and edited with Molecular Evolutionary Genetics Analysis Version 6.0 (MEGA6) ..37].Furthermore, the NA gene in this isolate contained arginine at positions 152 and 292 and histidine at position 274, indicating susceptibility to the NA inhibitors oseltamivir and zanamivir, which is similar to other H11N9 strains in and near Korea as well as A/swine/KU/2/2001(H11N6) .Mutations in internal genes which may increase replication efficiency as well as the virulence of a virus in different hosts indicate that H11N9 is virulent in avian and mammals as observed for previous H11N9 strains .As the genetic information implied that novel reassortment of H11N9 could increase replication efficiency, the viral replication of H11N9 was tested in vitro.50, plaque assay, and/or quantitative reverse transcription PCR with mathematical modelling are powerful approaches for performing detailed characterization of viral replication in vitro [Analysis of the TCIDin vitro .50 assay using the ELISA method to determine the virus-positive cells as previously described [50 assay are shown in Monitoring of influenza virus shedding is important in the investigation of virus adaptation to mammalian host. The human-origin virus, A/California/04/2009 (H1N1), which was donated from Korea National Institute of Health , was used as a control. DCK cells were infected with virus (MOI = 0.001). At every 12 hours, supernatants were collected and conducted for a TCIDescribed . The H1Nescribed  showed t5 EID50/50 μL of each virus was used to inoculate five 6-week-old female BALB/c mice. The infected mice displayed no severe clinical signs, including ruffled fur, depression, labored breathing and severe weight loss during 14 days postinfection (dpi) (50/mL at 3 dpi and absent in lung at 6 and 14 dpi while H1N1 maintained a comparatively high titer at 6 dpi (4.94 ± 0.18 log10 TICD50/mL)  a. All mion (dpi) b. The kiCD50/mL) . In 2014–2015, A/duck/Kagoshima virus was reassorted with subtype H11N9 viruses from Japan to produce an unknown A/Avian/ Kagoshima/2017(H11N9) with additional reassortment with other subtypes in 2015–2017. Until late 2017, unknown A/ Avian/ Kagoshima/2017(H11N9) circulated in eastern China and South Korea. In 2018, unknown A/ Avian/ Kagoshima/2017(H11N9) reassorted with PB1 of A/wild bird/Eastern China/1754/2017(H5N3) and PA of A/duck/Jiangsu/SE0261/2018(H5N3) to generate A/Mandarin duck/South Korea/KNU18-12/2018(H11N9) . Reassortment among different AIVs is the main mechanism involved in the generation of new types, which may increase mammalian transmission and threaten public health . In KoreCompared to imported strains from Russia–Mongolia-China to Korea, there are a few reports of genetic similarity between Japan- and Korea-derived AIVs ; mountaiH11N9 has been found in wild birds in Brazil, Europe, and Japan ,22,69,70All three strains isolated from Brazil consistently clustered together within evolutionary lineages of AIVs that had been previously described in aquatic birds in North America. Particularly, the H11N9 isolates were remarkably closely related to AIV strains from shorebirds sampled at the Delaware Bay region, on the Northeastern coast of the USA, more than 5000 km away from where the isolates were retrieved. Additionally, there was evidence of genetic similarity to AIV strains from ducks and teals from the interior USA and Canada . Our isoThe H11N9 strain analyzed in this study consistently clustered together within evolutionary branches of AIVs previously described in migratory birds in Japan. Six internal genes were closely related to AIV strains identified in migratory birds in Japan. The only exceptions were the PB1 and PA sequences, which were closely related to the Chinese AIV strains. PB1 clustered into Chinese strains between 2017 and 2018. Although substantial genetic similarity of PA was observed in A/duck/Jiangsu/SE0261/2018 (isolated in January 2018), it clustered with Japanese strains between 2015 and 2017 in phylogenetic analysis.According to the phylogenetic tree of HA and NA, the newly identified H11N9 clustered with the same strains as the most genetically similar strain (A/A/duck/Kagoshima/KU57/2014 (H11N9) rather than with Korean H11N9 found in 2016 and 2014–2015; dead spot-billed ducks carried two AIVs of the H11N9 subtype in the Izumi plain . As spotFurthermore, our H11N9 had a lower in vitro titer in MDCK cells than A/California/04/2009 (H1N1), supporting the LPAI characterization of H11N9. Although newly found H11N9 in 2018 exhibited low pathogenicity in mice, maintaining a database for H11N9 through careful surveillance is important because of its wide host tropisms (swine pig as host) and relatedness to H11N9 to contribute to the genesis of HPAI H7N9. 4.67 TCID50/mL) of A/California/04/2009 (H1N1) virus to mice in this study  was observed in lung of mice by 6 dpi, indicating the lower mouse adaptation of H11N9 than A/California/04/2009 (H1N1). A/California/04/2009 (H1N1)-infected mice survived up to 14 dpi without change of body weight, which corresponds to other previous reports ,73. It mis study . Mouse ais study . Satellite-tracking data supported that the crane used an eastern migration route from the Izumi plain in Japan followed the west coast of South Korea in the spring, moved to China for breeding in summer, and moved down to South Korea in autumn, after which it returned to the Izumi plain in Japan in winter in 2014–2016 .Interestingly, not only the H11 HA and N9 NA genes but also our isolate showed high sequence similarity to the corresponding genes of isolates from wild birds in South Africa and Spain, respectively, rather than those from East Asia, supporting that our isolate is related to the Japanese strains. The closest strains to A/Mandarin duck/South Korea/KNU18-12/2018(H11N9) HA were A/crane/Kagoshima/KU-T40/2015(H11N9) and A/duck/Kagoshima/KU57/2014(H11N9), showing HA from South Africa. These strains were closely related to A/red-billed teal/South Africa/KZN002/2012 (H11N2). The closest strain to A/Mandarin duck/South Korea/KNU18-12/2018 (H11N9) NA was A/crane/Kagoshima/KU-T40/2015 (H11N9) and A/duck/Kagoshima/KU57/2014 (H11N9), which originated from A/Anas crecca/Spain/1460/2008 H7N9. These results correspond to the previously identified H11N9 in the Izumi plain in Kagoshima, Japan . Wild aquatic birds such as dabbling ducks, gulls, and other shorebirds are considered as natural reservoirs for AIVs . H11N9 hH5 subtypes have mainly been found in Mandarin ducks. However, the susceptibility of Mandarin ducks to AIVs differs even for highly pathogenic AIVs depending on the genotypes. Mandarin ducks have not been shown to be susceptible to infection with Clade 2.3.4.4 Group B H5N8 and Group C H5N6 viruses . In contRecently, A/duck/Jiangsu/SE0261/2018 (H5N3) was isolated from domestic ducks in a poultry market in Jiangsu Province in the spring of 2018 .Interestingly, A/Mandarin duck/South Korea/KNU18-12/2018(H11N9) showed the highest PA gene similarity with A/duck/Jiangsu/SE0261/2018 (H5N3). Similarly, the other different subtype isolate (H2N9)  isolatedWe isolated a novel H11N9 in 2018 which was genetically different from previously reported Korean H11N9 in 2016. Although this research was based on the analysis of limited genetic diversity, the most likely route of infection of A/Mandarin duck/South Korea/KNU18-12/2018(H11N9) may be a reassortant virus shed by wild migratory birds in Japan and China to Korea.


CYP27B1 gene. CYP27B1 encodes an enzyme of 25-hydroxyvitamin D-1α-hydroxylase for converting inactive 25-OHD to biologically active 1,25-(OH)2D.Vitamin D-dependent rickets type 1A (VDDR1A) is a rare autosomal recessively inherited disorder due to loss-of-function mutations in the To identify underlying genetic defects in patients with VDDR1A.CYP27B1 gene were amplified by Polymerase Chain Reaction (PCR) from peripheral lymphocyte DNA. PCR products were directly sequenced. The consequences of c.590G > A mutation were analyzed by in silico and functional analysis.Twelve patients from 7 Turkish and 2 Saudi families were investigated. The coding exons and intron-exon boundaries of the CYP27B1 mutations were identified in all the patients. Two novel mutations were identified in two separate families: c.171delG (family 7) and c.398_400dupAAT (family 8). The intra-exon deletion of c.171delG resulted in a frameshift and premature stop codon 20 amino acids downstream from the mutation (p.L58Cfs∗20). The intra-exon duplication of c.398_400dupAAT generated a premature stop codon at the mutation site (p.W134∗). A missense c.590G > A (p.G197D) mutation was found in a patient from family 4 and caused a defect in pre-mRNA splicing. As a result, two populations of transcripts were detected: the majority of them with intron 3 retention (83%), and the minority (17%) being properly spliced transcripts with about 16% of wild-type enzymatic activity. The remaining nine patients from six families carried a previously reported c.1319_1325dupCCCACCC (F443Pfs∗24) mutation. Clinically, all the patients need continued calcitriol treatment, which was consistent with inactivation of 25-hydroxy vitamin D1α-hydroxylase activity.CYP27B1 mutations were identified and predicted to inactivate 25-hydroxyvitamin D-1α-hydroxylase. The loss of enzymatic activity by c.590G > A missense mutation was mainly caused by aberrant pre-mRNA splicing.Two novel frameshift  CYP27B1 gene (MIM 609506) and CYP2R1 gene (MIM 608713), respectively  and vitamin D-dependent rickets type 1B . VDDR1A and VDDR1B are caused by inactivation mutations in the ectively .2D by 25-hydroxyvitamin D-1α-hydroxylase. The biologically active 1,25(OH)2D binds to and activate vitamin D receptor to regulate calcium homeostasis and bone metabolism  by 25-hydroxylase (CYP2R1) . The 25-tabolism .2D despite normal or increased serum 25OHD um 25OHD . Based oc groups .In the present study, we investigated 12 VDDR1A patients from seven Turkish and two Saudi families.2D3 and elevated PTH. All of them required continued calcitriol treatment. The study was approved by the Institutional Review Board of King Faisal Specialist Hospital and Research Center and informed consent was signed by the guardian of the patients before enrollment.Twelve patients from seven Turkish (Family 1–7) and two Saudi families (Family 8–9) were enrolled for the study. Their clinical and laboratory findings were summarized in Genomic DNA was isolated from peripheral blood lymphocytes using the Gentra Blood Kit .CYP27B1 gene were amplified by PCR from genomic DNA as described previously . RT-PCR was performed to amplify the mini-gene transcripts using the following primer pairs: 5′-AACCCTGAACAACGTAGTCTG-3′ (in exon 3) and 5′-AACAGGAAGTGGGTCAGGTGC-3′ (in exon 5) for analysis of effect of c.590G > A mutation on pre-mRNA splicing. PCR conditions were 95°C for 5 min followed by 35 cycles of amplification  with final extension of 5 min. The PCR products were analyzed by agarose gel electrophoresis, and either directly sequenced or cloned into a TA vector  if multiple fragments were detected. Individual clones were subsequently sequenced.3  for 1 and 4 h, respectively. The 1,25(OH)2D3 in the medium was measured by radioimmunoassay (RIA) according to the manufacturer’s procedure .CHO cells were stably transfected with wild-type and c.590G > A mutant constructs as described previously . Cells wCYP27B1 gene.The clinical and biochemical features of patients were presented in CYP27B1 gene were sequenced from the patients and their parents. All the patients were homozygous for the CYP27B1 mutation and their normal parents were heterozygous carriers. Two novel mutations were identified: c.171delG in patient 10 from family 7 (∗20). The c.398_400dupAAT intra-exon duplication also created a premature stop codon at the mutation site (p.W134∗). The c.590G > A (p.G197D) mutation was found in patient 6 from family 4. Interestingly, the mutation was located at the first nucleotide of exon 4: at the junction of acceptor splice site of intron 3   .3, CHO cells transfected with wild-type CYP27B1 construct produced 285.6 ± 9.7 fmol/105 cells of 1,25(OH)2D3 as compared to 46.9 ± 2.8 fmol/105 cells transfected with G197D mutant. Thus, about 16% enzymatic activity were retained in the G197D mutant. Clinically, the patient responded to treatment very well. Initially, her weight and height standard deviation score (SDS) were −2.81 and −2.73, respectively. After treatment with calcitriol (60 ng/kg/day) and phosphate (55 mg/kg/day), her weight and height SDS became 1.42 and −0.45, respectively with normal serum Ca (9.5 mg/dL) and P (4.8 mg/dL). She is 9 years old now and currently under treatment with reduced dosage of calcitriol (16 ng/kg/day) and phosphate (9 mg/kg/day). Her last measurement of serum 25OHD3 was 42 ng/mL and 1,25(OH)2D3 was 42.1 pg/mL although serum Alp (380 IU) and PTH (125 pg/mL) were still elevated. The other patients also responded to treatment well but not as good as this patient.Although c.590G > A was previously reported , the funCYP27B1 mini-gene transcripts. Two populations of mutant mini-gene transcripts were detected: Mut-1 (83%) and Mut-2 (17%)  mutation, the translated protein have significantly reduced enzymatic activity.We next investigated effect of c.590G > A on RNA splicing. As shown in -2 (17%) . The intCYP27B1 inactivation and autosomal recessive inheritance.In the current study, we have identified two novel mutations (c.171delG and c.398_400dupAAT) and two previously reported mutations (c.590G > A and c.1319_1325dupCCCACCC). The loss of enzymatic activity by c.590G > A missense mutation is mainly mediated by pre-mRNA splicing defect. Furthermore, c.1319_1325dupCCCACCC is found in six out of nine families, the most frequent mutation encountered in the study. The patients’ clinical and genetic features are consistent with CYP27B1 gene  was found at the junction of donor splice-site (the last nucleotide of exon 7) (The c.171delG and c.398_400dupAAT frameshift mutations have not been reported in other populations and they may be unique in the Turkish–Arab populations. The c.1319_1325dupCCCACCC has been reported in other ethnic groups and is most common insertion mutations found in the 7B1 gene . Unlike  exon 7) . Althoug60G80| G99T99R95A71G81T46 . Guanine (G) at the last nucleotide of each exon occurs at 80% whereas A occurs at 9%, C 3%, and T 7%, respectively. The consensus sequences and base-level frequencies for acceptor splice-site (3′ splice-site) are C71A99G99| G52 (intron| exon). Guanine (G) at the first nucleotide of each exon occurs at 52% whereas A occurs at 24%, C 14%, and T 10%, respectively. The frequent appearance of G either at the last or first nucleotide of each exon indicates that G is relatively conserved at both 5′ and 3′ splice-sites. Given that c.590G is located at the first nucleotide of exon 4 (3′ splice-site) and is conserved, the G > A mutation would likely disrupt the recognition by the splice machinery to identify the correct acceptor splice site, resulting in intron 3 retention. On the other hand, the c.1215T is located at the last nucleotide of exon 7 (5′ splice-site) and is not conserved (G is the conserved nucleotide). The T > C mutation may not affect the recognition or base pairing between donor splice site of intron 7 and 5′ terminus of the U1snRNP in the early steps of splicing, thus having no effect on pre-mRNA splicing (CYP27B1 mutation.It has been reported that splice site recognition depends not only on its intrinsic strength (consensus vs. non-consensus sequences) but also on its context . Exon sesplicing . Althougsplicing , to our Splice-site mutations or intra-exon insertion/deletions such as c.171delG, c.398_400dupAAT, and c.1319_1325dupCCCACCC often result in a shift of the open reading frame, leading to a premature stop codon and truncated proteins devoid of function or degradation of transcripts due to non-sense-mediated decay . In bothCYP27B1 gene, which are predicted to inactivate CYP27B1 gene. The c.590G > A missense mutation impairs pre-mRNA splicing.In conclusion, we have identified two novel frameshift mutations in the The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Office of Research Affairs, King Faisal Specialist Hospital and Research Center. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.MZ and YS designed and analyzed the results and wrote the manuscript. AG and AA were involved in recruiting patients. HB, RA-R, MZ, BM, and AA were involved in acquiring data. 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.


Direct-seeded rice (DSR) is a potential technology for sustainable rice farming as it saves water and labor. However, higher incidence of weed under DSR limits productivity. Therefore, there is a need to develop herbicide tolerant (HT) rice varieties.AHAS) gene, which confers tolerance to imidazolinone group of herbicides from the donor parent (DP) “Robin” into the genetic background of an elite popular Basmati rice variety, Pusa Basmati 1121 (PB 1121). Foreground selection was done using the AHAS gene linked Simple Sequence Repeat (SSR) marker RM6844 and background selection was performed using 112 genome-wide SSR markers polymorphic between PB 1121 and Robin. Phenotypic selection for agronomic, Basmati grain and cooking quality traits in each generation was carried out to improve the recovery of recurrent parent phenome (RPP). Finally, a set of 12 BC4F4 near isogenic lines (NILs), with recurrent parent genome (RPG) recovery ranging from 98.66 to 99.55% were developed and evaluated. PB 1121-HT NILs namely 1979-14-7-33-99-10, 1979-14-7-33-99-15 and 1979-14-7-33-99-66 were found superior to PB 1121 in yield with comparable grain and cooking quality traits and herbicide tolerance similar to Robin.We used marker assisted backcross breeding (MABB) to transfer a mutant allele of Acetohydroxy acid synthase (AHAS allele. This is the first report on the development of HT Basmati rice. Superior NILs are being evaluated in the national Basmati trials, the release of which is likely to provide a viable option for the adoption of DSR technology in Basmati rice cultivation.Overall, the present study reports successful development of HT NILs in the genetic background of popular Basmati rice variety, PB 1121 by introgression of mutated  AHAS gene  for the transfer of HT trait. The mutant Robin was developed from an upland rice variety Nagina 22 (N22) through induced mutagenesis using ethyl methane sulfonate (EMS)  was backcrossed to PB 1121 to generate the BC2F1 seeds. A similar strategy was employed till BC4F1 generation, wherein in each of the generations, the plant heterozygous for the mutant AHAS allele along with maximum recovery for RPG and RPP were identified. The superior BC4F1 plants were advanced to BC4F2 generation and plants homozygous for the mutant AHAS allele were identified. Further, the selected BC4F2 plants were advanced to BC4F4 generation via pedigree-based phenotypic selection  method Doyle . The PCRAHAS allele was carried out using SSR marker RM6844 linked with AHAS gene at a distance of 1.2 cM in chromosome 2 . A total of 856 SSR markers were used to identify 112 polymorphic SSR markers between the parents PB 1121 and Robin, for use in background selection  Version 2.0 software  with three replications and sprayed with herbicide, Imazethapyr  @ concentration of 2.5 ml/liter, after 10 days of transplanting. Another set of same experimental material was grown side by side and used as unsprayed control with manual weeding. Visual observation of herbicide tolerance of the HT-NILs was made on 15 days after spray as per the standard protocol in rice , plant height (PH), number of productive tillers per plant (NPT), panicle length (PL), spikelet fertility percent (SF %), thousand grain weight (TGW) were recorded on five plants taken at random from the two middle rows of each plot under both sprayed and unsprayed conditions. The plot yield was recorded in kilogram/hectare (kg/ha) from each replication. The data on grain and cooking quality traits such as hulling percentage (HUL%), milling percentage (MIL%), head rice recovery percentage (HRR%), kernel length before cooking (KLBC), kernel breadth before cooking (KBBC), kernel length after cooking (KLAC), kernel breadth after cooking (KBAC), kernel elongation ratio (ER), alkali spreading value (ASV)  Little  and arom) Little  was recoCropStat 7.2Student’s t-test was performed for statistical significance differences for yield between unsprayed and herbicide sprayed condition at P ≤ 0.05.The statistical analysis of agro-morphological data was carried out using CropStat 7.2  and with a higher level of phenotypic similarity to PB 1121 was backcrossed and 188 BC2F1 plants were generated, of which 94 plants were heterozygous for foreground marker, RM6844 and the RPG recovery ranged from 81.25 to 92.85%. The superior BC2F1 plant was further backcrossed and 152 BC3F1 seeds were produced, out of which 73 plants were found to be heterozygous for the foreground marker and these plants were subjected to phenotypic evaluation for agro-morphological traits and background selection was done using 16 SSR markers remained heterozygous in the BC2F1 generation. The RPG recovery in the BC3F1 generation ranged from 93.30 to 95.08%. One superior BC3F1 plant, with maximum RPG (95.08%) and relatively superior recovery for RPP was backcrossed to generate 122 BC4F1 plants. A total of 58 plants were found to be heterozygous for RM6844 with RPG recovery range from 94.64 to 98.21%. A plant with maximum RPG recovery (98.21%) was selfed to produce BC4F2 population. Out of 384 BC4F2 plants, 88 plants were found to be homozygous for AHAS gene linked SSR marker RM6844  and high volume expansion upon cooking (> 4 times)  in aromatic rice is explained by the loss of function mutations in the badh2 gene  of all 112 SSR markers polymorphic between RP PB 1121 and DP Robin. M: 50 base pair DNA ladder; P1: PB 1121; P2: Robin.Additional file 2: Figure S2. Chromosome wise Graphical representation of all 112 SSR markers polymorphic between RP PB 1121 and DP Robin.Additional file 3: Figure S3. Gel image showing the amplification profile of AHAS linked SSR marker RM6844 in the F1 plants. M: 50 base pair DNA ladder; DP: Robin; RP: PB 1121; 1–15: F1 plants.Additional file 4: Figure S4. A representative gel amplification image of the SSR marker, RM6844 used in foreground selection in the BC4F2 population. M: 50 base pair DNA ladder; RP: PB 1121; DP: Robin; 1–68: BC4F2 plants.Additional file 5: Table S1. Details of 112 polymorphic SSR markers used in Background selection for development of PB 1121 HT-NILs.


Arabidopsis thaliana, members of the uridine diphosphate-dependent glucosyltransferases UGT72E and UGT72B subfamilies have been demonstrated to glycosylate monolignols. Here, the poplar UGT72 family, which is clustered into four groups, was characterized: Group 1 UGT72AZ1 and UGT72AZ2, homologs of Arabidopsis UGT72E1-3, as well as group 4 UGT72B37 and UGT72B39, homologs of Arabidopsis UGT72B1-3, glycosylate monolignols. In addition, promoter-GUS analyses indicated that poplar UGT72 members are expressed within vascular tissues. At the subcellular level, poplar UGT72s belonging to group 1 and group 4 were found to be associated with the nucleus and the endoplasmic reticulum. However, UGT72A2, belonging to group 2, was localized in bodies associated with chloroplasts, as well as possibly in chloroplasts. These results show a partial conservation of substrate recognition between Arabidopsis and poplar homologs, as well as divergent functions between different groups of the UGT72 family, for which the substrates remain unknown.Monolignols are the building blocks for lignin polymerization in the apoplastic domain. Monolignol biosynthesis, transport, storage, glycosylation, and deglycosylation are the main biological processes partaking in their homeostasis. In  Arabidopsis thaliana and Populus trichocarpa  in P. trichocarpa [Glycosyltransferases (GT) EC 2.4.x.y) are defined as enzymes that utilize an activated donor sugar substrate that contains a (substituted) phosphate leaving group . Glycosy.4.x.y arses.html . The numchocarpa . UGTs gl1 out of 88 GT in p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol, are the main building blocks of lignin and (neo)lignans [p-coumaryl alcohol glucoside, coniferin, and syringin, respectively (Monolignols (or (hydroxyl)cinnamyl alcohols), i.e., )lignans . When glectively . Monoligp-coumaryl alcohol plasma membrane transporter, AtABCG29, has been identified and characterized in Arabidopsis [Glycosylation of monolignols renders them less cytotoxic by reducing their reactivity . Monoligbidopsis . Howeverbidopsis  by molecO glucosylate coniferaldehyde and sinapaldehyde, as well as coniferyl and sinapyl alcohols with different specificities. UGT72E2 and UGT72E3 also accept ferulic and sinapic acids as substrates [p-coumaryl, coniferyl and dihydroconiferyl alcohols, coniferaldehyde, and p-coumaraldehyde, while UGT72B3 could glucosylate coniferaldehyde and sinapaldehyde [O-glucoside and sinapic acid 4-O-glucoside, was measured in Arabidopsis overexpressing UGT72E genes, with specificities depending on the gene overexpressed [UGT72E1-3, for which no lignin-associated phenotype and/or lignin modification  was reported, the ugt72b1 mutant was characterized by an ectopic lignification in floral stems, along with arrested growth [ugt72b1 mutant and transgenic Arabidopsis lines overexpressing UGT72B1 had an increase in coniferin [ugt72b1 mutant is possibly related to the increased expression of both UGT72B3 and UGT72E2 that was detected in this mutant.In Arabidopsis, several recombinant proteins belonging to the UGT72 family, including UGT72E1-3 ,29, as waldehyde . In contaldehyde ,30. An axpressed . In contOverall, these reports indicate that different recombinant glycosyltransferases from the Arabidopsis UGT72 family have different substrate specificities in vitro, suggesting that they are all involved in a complex regulatory network required for adequate maintenance of the monolignol/monolignol glucoside pool, as well as that of their acid or aldehyde precursors .P. tremula × P. alba clone INRA 717-1B4), a woody perennial plant, to identify which members are involved in the glycosylation of monolignols. Our results showed that poplar UGT72 family members clustered into four groups. Enzymatic activities of each member, tested with 11 different phenylpropanoid substrates, as well as analysis of glycosylated monolignols in transgenic poplars overexpressing the corresponding genes, allowed the identification of 4 poplar UGT72s glycosylating monolignols, including members of group 1 (UGT72AZ1 and UGT72AZ2) and group 4 (UGT72B37 and UGT72B39). The poplar UGT72s belonging to group 2 and 3 had no activity towards the tested substrates. Analysis of promoter-GUS expression profiles showed expression in vascular tissues for all genes analyzed. Finally, differences in subcellular localization were found for the poplar UGT72s. Altogether, these data suggest different functions for members of the UGT72 family in poplar.The aim of the present study was to characterize the UGT72 family in poplar , and Potri.014G041900 was not cloned as it has a truncated PSPG motif, and the catalytically active His at the N-terminus is missing . An alignment of the different deduced polypeptide sequences of UGT72 members of P. tremula × P. alba and Arabidopsis shows the PSPG domain and conserved key residues in these sequences  tag. After production in E. coli and purification, recombinant proteins were tested using 11 substrates of the phenylpropanoid/monolignol pathway  and UGT72BB1 (group 3) are expressed at low levels in leaves (transcript abundance between 2 and 8), whereas UGT72A2 (group 2) shows an intermediate expression (transcript abundance of approximately 150) compared to UGT72B36-39 (group 4), which are highly expressed (transcript abundance between 150 and 1000)  .UGT72 overexpressing lines. To further characterize these lines, we investigated the profiles of glycosylated phenolics in leaf extracts in order to identify the in vivo acceptor substrates. LC-MS analysis (UGT72AZ1), or just coniferin (UGT72AZ2). Traces of coniferin were detected in the WT, while syringin was below the detection level. Transgenic lines overexpressing group 2 or group 4 UGT72 genes did not accumulate detectable amounts of both glycosides using a HPLC-UV system .Examination of the transgenic plants revealed that no phenotypical (at the whole plant level), nor anatomical differences  could be observed in any of the different poplar analysis  indicateTo assess whether the observed increase in the quantity of coniferin and syringin levels in leaves was accompanied by modifications in lignin levels, we analyzed stem wood, stem bark, and root samples from 35S-UGT72AZ1 and 35S-UGT72AZ2 lines. Our results indicated that lignin content, as measured by the acetyl bromide method, was not affected in any of the samples from these lines .UGT72 genes, their expression profile was analyzed by RT-qPCR in different parts of the stem and the root of 4-month-old greenhouse grown WT poplar. Primary vascular tissues (primary VT) correspond to the first 2 cm below the apex. Secondary vascular tissues (secondary VT) were sampled 50 cm below the apex and separated into bark and wood , and UGT72B36 in xylem of the stem and in old roots (thus possibly in the xylem of root), while UGT72B37 was expressed in all parts with the highest level in stem secondary xylem, UGT72B38 in young stems and roots (thus possibly in primary VT), and UGT72B39 in the xylem of the stem and in young roots (probably in the xylem of the root).To further characterize the different poplar UGT72s spatial expression, histochemical GUS staining of pUGT72::GUS transgenic poplar lines was investigated. Unfortunately, no plants could be regenerated for the pUGT72B38::GUS and pUGT72BB1::GUS constructs. A focus was made on the organs where the expression measured by RT-qPCR of the respective genes was most pronounced . As shown in UGT72 genes are expressed in vascular tissues with different specificities. UGT72AZ1 is expressed mainly in the phloem within the stem and leaf , Secondary wall NAC-Binding Element (SNBE), R2R3MYB Responsive Element (AC-elements), and Tracheary-Element-Regulating cis-Element (TERE) [pUGT72B39-BB1 to five elements in pUGT72A2/AZ2). TERE is found in several genes related to xylem differentiation [pUGT72B36. Several SMRE and AC-elements were detected in pUGT72BB1, pUGT72B36, pUGT72B37, and pUGT72B39, while pUGT72B38, pUGT72AZ1, and pUGT72A2 displayed a single SMRE, and pUGT72AZ2 did not exhibit any of these cis-elements. The presence of these cis-elements may explain the preferential expression of the poplar UGT72 genes in vascular tissues  ,37. SNBEntiation  and was  tissues .P. tremula × P. alba grown in the greenhouse  and UGT72A2 (group 2), for which custom antibodies were designed, was investigated in 35S::UGT72A2-GFP construct. In this case, the localization of UGT72A2-GFP was also observed in bodies associated with the chloroplasts and possibly within chloroplasts  marker. As shown in roplasts g.UGT72 family in poplar, a woody perennial plant, and to identify genes that could be involved in the glycosylation of monolignols. UGTs are known to show in vitro activities towards a large range of different substrates, and their substrate specificity cannot be predicted merely based on phylogenetic relationships [UGT72 family, a phylogenetic tree of Arabidopsis and poplar UGT72 members was generated allowing the identification of 4 groups in poplar, of which 3 were common with Arabidopsis , no incGUS expression driven by the poplar UGT72 promoters was observed in the vascular tissues for the investigated genes  and genes involved in secondary cell wall formation [UGT72B promoters contain elements recognized by these transcription factors, therefore, suggests that they are part of the transcriptional programme leading to secondary cell wall formation in xylem. Besides, single or several SNBE were also found in pUGT72s. These elements are targeted by NAC transcription factors, in which downstream-regulated genes partially overlap with MYB-induced genes involved in secondary cell wall formation [CesA4 and CesA8 [XYLEM CYSTEINE PEPTIDASE 1 and 2 [METACASPASE 13 and 14 [cis-element is enriched in genes involved in tracheary element differentiation, comprising cell death [UGT72s may also play a role during the late phase of xylem differentiation. We performed a similar analysis on promoters of Arabidopsis UGT72s , lignin polymerization, as well as regulation of secondary cell wall formation, were upregulated in this mutant, indicating feedback regulation upon disturbance of monolignol glucosylation on whole lignin synthesis, by a mechanism that awaits further discovery [ugt72b1 mutant, the ectopic lignification phenotype is probably an indirect consequence of the mutation. This transcriptional shift is likely gene- and species-dependent, which may explain the absence of phenotype in lines overexpressing poplar UGT72 members  and group 4 (UGT72B36-37 and 39) were localized in the nucleus and associated with the ER . AccordiAgrobacterium, while, in stable transgenic lines carrying the same construct, the signal was also detected inside chloroplasts. These bodies are usually observed when large quantities of proteins are present, either due to high expression driven by the CaMV35S promoter or when high quantities of plasmids are used for the transfection [In contrast, UGT72A2 (group 2) was found to be associated with chloroplastic bodies, as well as possibly localized within chloroplasts, suggesting a different function than the other UGT72s from poplar. More precisely, UGT72A2-GFP was localized in bodies associated with the chloroplast following tobacco transient transformation with sfection ,66. We msfection  in poplaPopulus tremula × P. alba, clone INRA 717-1B4) were grown aseptically or in the greenhouse, as described previously [Both WT and transgenic poplars . Ten polypeptides sharing ~ 45% identity with Arabidopsis UGT were identified. P. trichocarpa coding sequences were then used to identify corresponding P. tremula × P. alba coding sequences using BLASTN in the P. tremula × P. alba clone INRA 717-1B4 genome sequence (https://urgi.versailles.inra.fr/Species/Forest-trees/Populus/Clone-INRA-717-1B4). P. tremula × P. alba gene specific primers were designed using PerlPrimer (v1.1.21)  and sequenced. Cis-elements related to secondary cell wall formation were retrieved from the promoter 1500 bp upstream of the start codon with QIAGEN CLC Genomics Workbench (v20) and visualized online with IBS [Arabidopsis UGT72s protein sequences were used to screen the v1.1.21)  to ampliwith IBS .Full-length protein sequence alignment (MUSCLE) and generation of the phylogenetic tree using the approximate likelihood-ratio test (SH-like) in PhyML was performed online .P. tremula × P. alba  grown in the greenhouse using the Agilent Plant RNA isolation mini kit . Within the stem, three samples were harvested. The first one corresponded to the 2 first cm of the stem, including the apex and leaf primordia, corresponding to primary VT. For the second and third samples, a portion of the stem was removed at 50 cm below the apex (corresponding to secondary VT) and separated into bark (phloem) and wood (xylem). For the roots, primary VT corresponded to young root with primary structure, and secondary VT corresponded to roots with a diameter of 1 cm. The reverse transcription was carried out with GoScript Reverse Transcription Mix, Oligo(dT) kit . The qPCR was carried out with Luna Universal qPCR Master Mix and the LightCycler 480 system  in technical triplicate. Amplification specificity was checked at the end of each qPCR run with a melt curve. The obtained values were normalized with two reference genes . The gehttp://www.thermoscientific.com) and observed under a light microscope.Histochemical GUS staining was performed as described by Hemerly et al.  with modE. coli BL21(DE3) strain in the vector pBAD-DEST49  (Odyssey Fc Imaging System (LI-COR Biosciences). The image processing was realized with Image Studio Lite (LI-COR biosciences).Polyclonal antibodies raised against specific peptides of UGT72AZ1 (TDNSLSKSQLLKQSPC), UGT72AZ2 (CKSVRFEDTLPAYLDR), and UGT72A2 (LTRPLKPLDSRSGEL) were produced and quality-checked externally . The cross-reactivity of the purified antibodies was tested by immunoblot with the recombinant proteins UGT72AZ1, AZ2, and A2 produced in is, USA) . The expFor immunolocalization, stem pieces were fixed with a solution of 4% formaldehyde and 0.1% Triton in 50 mM PIPES overnight at 4 °C with agitation after a vacuum for 30 min. After fixation, stem sections (100 µm) obtained using an HM 650 V vibratome were incubated with primary antibodies  overnight at 4 °C. Sections were washed thrice with 50 mM PIPES buffer, incubated for 1 h 30 min at 37 °C with anti-rabbit antibodies conjugated to AlexaFluor 488  and washed thrice with 50 mM PIPES buffer before observation using a Zeiss confocal microscope LSM710. Secondary antibody was detected with the following parameters: excitation at 488 nm, emission recorded between 495 and 573 nm. Lignin autofluorescence was detected with the following parameters: excitation at 405 nm, emission recorded between 410 and 507 nm. All acquisition settings were similar for sections incubated with the different antibodies, and no change was made to image display post-acquisition.UGT72s were cloned in the vector pK7FWG2 [35S::AtWAK2–mCherry construct was used as marker for ER [A. tumefaciens (strain C58RifPMP90) by electroporation. After separated preculture in LB medium with 10 mM MES buffer and 20 µM acetosyringone (supplemented with the selection antibiotic), bacterial suspensions were mixed, and agroinfiltration were performed in tobacco leaves. After 2 to 4 days, leaves were analyzed with a LSM710 confocal microscope. The wavelength of excitation for GFP is 488 nm, and the emission was recorded between 495 and 543 nm. The wavelength of excitation for mCherry is 543 nm, and the emission was recorded between 560 and 630 nm. The analysis was not performed for UGT72BB1 and UGT72B38.Poplar  pK7FWG2  by Gatewr for ER . Each coE. coli BL21(DE3)pLysS genotype using UGT72s cloned into pGEX-4T-1 . Following transformation, a preculture grown at 37 °C was used to inoculate 500 mL of LB supplemented with ampicillin (100 µg/mL) and chloramphenicol (34 µg/mL). Induction with IPTG  was performed at an OD600 of 0.5–0.6, followed by overnight incubation at 18 °C under agitation (200 rpm). Cells were then pelleted, washed, and stored at −80 °C until protein purification. The pellet was then resuspended in 10 mL GST-Wash/Bind buffer supplemented with PMSF  and lysozyme and ultrasonicated. The resulting bacterial lysate was centrifuged , and the supernatant was applied to a column with GST•Bind™ resin (Novagen). GST-tagged proteins were allowed to bind to the resin overnight at 4 °C under soft agitation. After three washings, proteins were eluted in 5 consecutive fractions (300 µL each). The purity of these 5 fractions was investigated through SDS-PAGE, before pooling the pure fractions for quantification with the Roti-Nanoquant assay (Carl Roth).Recombinant protein production was performed in p-coumaraldehyde, sinapaldehyde, sinapyl alcohol, trans-cinnamic acid, p-coumaric acid, caffeic acid, ferulic acid, sinapic acid, and salicylic acid) in a total volume of 100 µL. Each reaction was performed in triplicate and incubated overnight at 30 °C. The reaction was stopped by heating at 75 °C for 10 min, centrifuged, and analyzed with a LC-UV-MS/MS system using the protocol described in Reference [The enzymatic assay was performed with 5 µg of purified protein and the following final concentrations: 100 mM Tris HCl pH7.5, 1 mM UDP-glucose, 600 µM substrate . Poplar transformation was made as described in [For the overexpression,  PK7WG2D  and prom PK7WG2D  by Gatewribed in . A minimLignin content was determined in cell wall residue  made froPlant material was ground in liquid nitrogen for each line . Metabolites were extracted on 200 mg (fresh weight) with 1 mL methanol 80% on ice over 4 h with several vortexing steps. After centrifugation, the extract was filtered on a 0.22 µm membrane, aliquoted, and 20 µL were injected in a HPLC-UV-MS system (Agilent 1260 Infinity II coupled to Advion Expression CMS L). Metabolites were separated using a water (0.1% formic acid)/acetonitrile gradient (10 to 30% ACN in 20 min) at 1 mL/minute on a C18 column . Identification of coniferin and syringin was based on their retention time, absorption at 264 nm and mass spectra by comparison with authentic standards. Differences in phenolic abundance between WT and overexpressing lines were assessed by comparing UV profiles at 264 nm.UGT72AZ1 and UGT72AZ2 overexpressing lines was measured through LC-MS analysis on an ACQUITY UPLC I-Class system , coupled to a Synapt QTOF hybrid mass spectrometer (Waters Corporation). Chromatographic separation was carried out on an ACQUITY UPLC BEH C18  column from Waters, temperature was maintained at 40 °C. A 30 min gradient of two buffers was used: buffer A (99/1/0.1 H2O/ACN/formic acid pH3), buffer B (99/1/0.1 ACN/H2O/formic acid pH3); 95% A for 0.1 min decreased to 50% A in 30 min. The flow rate was set to 0.35 mL/minute, and the injection volume was 15 μL. The MS LockSpray ion source was operated in negative electrospray ionization (ESI) mode under the following specific conditions: capillary voltage: 2.5 kV; source temperature; 120 °C; desolvation gas temperature; 400 °C; desolvation gas flow; 550 L/h. Mass range was set from 100–1000 Da. Nitrogen (>99.5%) was employed as desolvation and cone gas. Leucin-enkephalin (250 pg/μL solubilized in water/ACN 1:1 (v/v), with 0.1% formic acid) was used for the lock mass calibration. Centroided data was recorded through Masslynx software (Waters). Chromatogram processing and data normalization was performed with Progenesis QI software v2.1 (Waters). Database maintenance for compound identification was done with JChem for Excel add-in .Accumulation of coniferin and syringin in UGT72A2), MT181027 (UGT72AZ1), MT181028 (UGT72AZ2), MT181029 (UGT72B36), MT181030 (UGT72B37), MT181031 (UGT72B38), MT181032 (UGT72B39), MT181033 (UGT72BB1), MT193261 (pUGT72A2), MT193262 (pUGT72AZ1), MT193263 (pUGT72AZ2), MT193264 (pUGT72B36), MT193265 (pUGT72B37), MT193266 (pUGT72B38), MT193267 (pUGT72B39), and MT193268 (pUGT72BB1).Gene sequence data from this article are deposited in GenBank under the accession numbers MT181026 (UGT72s are At3G50740 (UGT72E1), At5G26310 (UGT72E2), At5G66690 (UGT72E3), At4G01070 (UGT72B1), At1g01390 (UGT72B2), At1g01420 (UGT72B3), At4G36770 (UGT72C1), At2G18570 (UGT72D1), and At2G18560 (UGT72D2P). The P. trichocarpa accession numbers corresponding to the cloned UGT72s from P. tremula × P. alba are Potri.007G030300 (UGT72AZ1), Potri.007G030400 (UGT72AZ2), Potri.007G030500 (UGT72A2), Potri.014G041800 (UGT72BB1), Potri.014G096000 (UGT72B36), Potri.014G096100 (UGT72B37), Potri.003G138200 (UGT72B38), and Potri.002G168600 (UGT72B39).The accession numbers of the Arabidopsis UGT72s, like their Arabidopsis counterparts, are expressed within vascular tissues. No direct impact of the overexpression of poplar UGT72s on lignification was evidenced. Finally, the observed differences in subcellular localization suggest divergent functions of members of the poplar UGT72 family. Further studies on poplar mutants and identification of substrates are required to unravel the role of UGT72s, particularly in the lignification process or in defense towards pathogens in poplar.The survey of the UGT72 family in poplar allowed the identification of members that are involved in the glycosylation of monolignols and that are clustered with Arabidopsis UGT72Es (group 1) and UGT72Bs (group 4). In addition, poplar