Paraburkholderia phymatum STM815, a rhizobial strain of the Burkholderiaceae family, is able to nodulate a broad range of legumes including the agriculturally important Phaseolus vulgaris (common bean). P. phymatum harbors two type VI Secretion Systems (T6SS-b and T6SS-3) in its genome that contribute to its high interbacterial competitiveness in vitro and in infecting the roots of several legumes. In this study, we show that P. phymatum T6SS-b is found in the genomes of several soil-dwelling plant symbionts and that its expression is induced by the presence of citrate and is higher at 20/28°C compared to 37°C. Conversely, T6SS-3 shows homologies to T6SS clusters found in several pathogenic Burkholderia strains, is more prominently expressed with succinate during stationary phase and at 37°C. In addition, T6SS-b expression was activated in the presence of germinated seeds as well as in P. vulgaris and Mimosa pudica root nodules. Phenotypic analysis of selected deletion mutant strains suggested a role of T6SS-b in motility but not at later stages of the interaction with legumes. In contrast, the T6SS-3 mutant was not affected in any of the free-living and symbiotic phenotypes examined. Thus, P. phymatum T6SS-b is potentially important for the early infection step in the symbiosis with legumes. Competi legumes . This higenotype . In othegenotype , motilitgenotype , and catgenotype  have beepetition . The T6Sbacteria . Interesfect pea . The numfect pea . The corfect pea . The memfect pea . The tubfect pea . The purty genes . The effty genes . These ety genes . Only revulgaris . HoweverP. phymatum T6SS-b and T6SS-3 clusters in vitro in response to different carbon sources and temperatures. Furthermore, we explored the expression and role of the T6SS clusters during symbiosis, in the presence of germinated seeds and within root nodules. We show that T6SS-b is also present in other soil bacteria and its expression is elevated at lower temperature and in the presence of citrate as carbon source. In contrast, T6SS-3 is also found in pathogenic Burkholderia strains , which may explain its higher expression at 37°C and in stationary phase. Moreover, T6SS-b was also expressed in the presence of germinated seedlings and in P. vulgaris and Mimosa pudica root nodules. Phenotypical analysis indicated a role of T6SS-b in motility, suggesting that this cluster may play a role in the early step of plant infection.In this study, we first evaluated the phylogenetic conservation and expression of the two Paraburkholderia strains were grown in Luria-Bertani (LB) medium without salt at 28°C and 180 rpm (Escherichia coli and 80 μg/ml for P. phymatum), kanamycin , tetracycline , trimethoprim . For the induction of the promotors fusions in AB minimal medium  and p6115 (upstream of tssH in T6SS-3) were constructed using the vector pPROBE-NT as previously described (nodB), were PCR amplified by using phymatum STM815 genomic DNA (gDNA)  and Bphy_5979 (tssC), which builds the sheath of the T6SS-b, were deleted. The primers Bphy_5978_up_F_NotI and Bphy_5978_up_R_MfeI were used to amplify the upstream fragment and the primers Bphy_5979_dn_F_NdeI and Bphy_5979_dn_R_NotI for the downstream fragment. In between the two fragments, was cloned a trimethoprim resistance cassette dhfr with a transcription terminator which was amplified from plasmid p34E-TpTer (dhfr) or a chloramphenicol cassette (catA2) cloned from plasmid pSHAFT with the primers catA1_F_EcoR1 and catA2_R_NdeI. The Bphy_6113 upstream fragment was amplified with the primers Bphy_6114_up_F_XbaI and Bphy_6114_up_R_MfeI. The Bphy_6114 downstream fragment was amplified from the gDNA with the primers Bphy_6113_dn_F_NdeI and Bphy_6113_dn_R_XbaI. The ligated inserts were cloned into suicide plasmids pSHAFT and pEX18-Tc, respectively, resulting in constructs pSHAFT::ΔT6SS-3 and pEX18-Tc::ΔT6SS-3. The deletion mutant STM815-ΔT6SS-3 (dhfr) was chloramphenicol sensitive and trimethoprim resistant, mutant STM815-ΔT6SS-3 (catA2) was tetracycline sensitive and chloramphenicol resistant. To construct a ΔΔT6SS mutant, the plasmid pEX18-Tc::ΔT6SS-3 (dhfr) was transferred by triparental mating in P. phymatum ΔT6SS-b. The resulting mutant strain was named P. phymatum STM815 ΔΔT6SS.The two promotor fusions p5978 (upstream of escribed . The proA (gDNA)  with priA (gDNA) . The clo4E-TpTer  with the600). The GFP expression of bacteria in solid media were visualized by a digital camera  with GFP filter. The different T6SS reporter constructs  were grown with different carbon sources in 96-well plates  at 30°C for 48 h. Two biological replicates of each strain were tested. To observe the temperature dependent GFP expression of each promoter reporter, bacteria were grown in 50 ml LB-NaCl in 250 ml Erlenmeyer flasks (starting OD600 = 0.05) at 180 rpm at different temperatures . To visualize the GFP expression responding to the roots of bean germinated seedlings, a suspension of the P. phymatum reporter strains  was mixed with melted soft agar (0.8%) ABS medium. A germinated bean was placed in the middle of the plate with root tip stabbed into the soft agar medium. After 3 days of incubation at 30°C, the primary roots were inspected under a Leica M165 FC fluorescent stereo microscope, and their relative fluorescent signal acquired through a GFP2 filter set  at constant focal distance and acquisition settings . The green channel of resulting RGB images was separated in FiJi and the mean gray signal, 1 mm around each root apex was quantified. The expression of GFP in the nodules of bean and mimosa inoculated with the P. phymatum reporter strains  was quantified by using a Leica SPE DM5500Q confocal LASER scanning microscope with an ACS APO 40.0× 1.15 oil objective and the images were analyzed with Fiji  with excitation at 488 nm and emission at 520 nm, recording fluorescence in relative fluorescence units (RFU) and the cell density  and discs containing 10M, 5M, 1M H2O2, and H2O were placed on the plate and images were taken after 24 h incubation.The swimming motility assay was carried out as previously described  in a difP. vulgaris, cv. Negro Jamapa) and mimosa seeds (M. pudica) were surface sterilized as previously described (600 = 0.025 (107 cells pro ml) were inoculated onto each seed and the plants were incubated for 21 (bean) or 28 (mimosa) days in the green house . The colony forming units (CFU) of each inoculum was determined on LB-NaCl plates. The plants were watered twice a week with autoclaved deionized H2O. Symbiotic properties  were determined as previously described  was searched and sequences of the retrieved homologous clusters were searched again to identify additional, more distantly related clusters. Since the T6SS-3 cluster consisted of two operons pointing in opposite directions  and MEGA X was used to construct a maximum likelihood phylogenetic tree with a Tamura-Nei parameter  .To find homologs in other bacteria, the n 2.9.0+  against rections , the seqlustalX2  was usedp = 100) . PutativP. phymatum STM815 genome (tssHBCDIKLMI and the other (from Bphy_6116 to Bphy_RS30580) carrying tssEFGA and tssI  in the T6SS-b cluster, we found eight genes coding for putative proteins: an YwqK family antitoxin (Bphy_5986), a PAAR-like protein (Bphy_5987), a hypothetical protein (HP) (Bphy_5988), an amino acid ABC transporter (Bphy_5989), a sodium:dicarboxylate symporter (Bphy_5990), an aspartate/glutamate racemase (Bphy_5991) and a second sodium:dicarboxylate symporter (Bphy_5992) . An alignment of the protein sequences of these different dicarboxylate symporters suggests that the symporters located in T6SS-b likely transport a different substrate compared to that of the known DctA transporters (vgrG (Bphy_6124) located in the T6SS-3 cluster, five genes potentially coding for effectors were identified: two M23 family metallopeptidases (Bphy_6125 and Bphy_RS30575), Bphy_6126 and Bphy_6128 both coding for a HP and Bphy_6127 coding for a PAAR-like protein . We next looked for the presence of similar T6SS clusters in other strains by blasting the DNA sequence of either the entire cluster for T66S-b, i.e., from Bphy_5978 (tssB) to Bphy_5997 (tssA), and of the two operons of T6SS-3, i.e., from Bphy_6107 (tssL) to Bphy_5107 (tssH) and Bphy_6116 to Bphy_RS30575 against the NCBI nt database3 . Fifteen strains were found to contain a T6SS-b cluster similar to that of P. phymatum STM815. These strains exclusively represent beneficial bacteria occurring in the soil; they include six Paraburkholderia strains , three Cupriavidus strains , Achromobacter insolitus FDAARGOS 88, Variovorax sp. PMC12, and Rhodoferax saidenbachensis DSM22694. One strain (Janthinobacterium sp. 1-2014MBL) had three additional genes downstream of vgrG, one coding for a GIY-YIG nuclease family protein and two encoding HPs. Moreover, the T6SS-b cluster in Janthinobacterium sp. 1-2014MBL did not contain the racemase encoding gene between the two sodium:dicarboxylate symporters. A phylogenetic analysis based on the DNA sequence of the clusters grouped the T6SS-b clusters of these bacteria in two clades  together with potential effector genes.Two T6SSs clusters have been identified in the 5 genome , which c5 genome . Accordi5 genome . While cand tssI . Each clhy_5992) . The presporters . Downstr protein . As in mty genes . P. phymo clades . In cont strains . In ordevgrG. To investigate if these transporters (Bphy_5990 and Bphy_5992) are involved in induction of T6SS-b expression, the reporter constructs for T6SS-b (p5978) and T6SS-3 (p6115 and p6116) , fumarate (26 h), citrate (36 h), and aspartate (38 h). T6SS-b (p5978) and T6SS-3 (p6115 and p6116) expression was followed for 48 h and in As mentioned above, the T6SS-b cluster contains two sodium:dicarboxylate symporters downstream of d p6116)  were groBurkholderia such as B. pseudomallei and B. mallei strains  up-regulated when P. phymatum was grown at lower temperatures (20 and 28°C). T6SS-b expression increased about 3.3-fold at 20°C compared to 37°C. In contrast, higher T6SS-3 expression levels were determined at 37 vs 28°C . Thus, it appears that both systems are subjected to temperature-dependent regulation, which seems to be in line with the different origin of both gene clusters  fused to gfp was used as a positive control. The gene nodB is involved in the biosynthesis of the backbone of the NF which is produced in response to flavonoids secreted by the root. Next, a 2 days-old germinated bean seed was added on the plate and the expression was analyzed under a fluorescence stereomicroscope after 3 days of incubation at 28°C. While the T6SS-b cluster (p5978) was expressed, at the tip of the root, both T6SS-3 reporter constructs p6115 and p6116 showed low expression in the presence of the germinated bean seed (nodB (p7722) was highly expressed displaying a diffused signal around the root, confirming the well-known induction of nodB expression by root exudates. Since T6SS-b expression was localized on the root, we suggest that T6SS-b expression is induced by a component present on the tip of the root.To assess the expression of ean seed . The GFPean seed . As expeP. phymatum T6SSs clusters during symbiosis in root nodules was analyzed using P. vulgaris and M. pudica as host plants. The plants were infected with the P. phymatum T6SS-b (p5978) and T6SS-3 (p6115 and p6116) reporter strains and the root nodules analyzed 21 or 28 dpi. The T6SS-b (p5978) was expressed at a higher level than T6SS-3 (p6115 and p6116) in P. vulgaris and M. pudica  in each T6SS cluster (ΔTssBC-b and ΔTssBC-3) and we constructed a T6SS double mutant strain (ΔΔTssBC). The growth profile of all T6SS mutants in LB without NaCl showed a slightly faster growth of the ΔΔT6SS, while the growth of the ΔT6SS-b and ΔT6SS-3 strains was similar to the growth of the wild-type. We tested phenotypes relevant in different steps of the rhizobium-legume symbiosis such as exopolysaccharide (EPS) production, motility, root attachment, and sensitivity to H2O2 and antibiotics (2O2) and to different antibiotics  compared to the wild-type (2O2 (data not shown). Next, the symbiotic properties of ΔT6SS-b, ΔT6SS-3, and ΔΔT6SS strains  were analyzed during symbiosis with P. vulgaris and M. pudica as host plants and compared with plants infected with P. phymatum wild-type  of R. leguminosarum was shown to be required to extend the host range of this rhizobium   or bacteria found in the root endosphere (P. hospita BT03 was found in Populus deltoides)  within the T6SS-b cluster, where usually effectors are located, we found two genes coding for potential sodium:dicarboxylate symporters  positively controls expression of T6SS-b under nitrogen limiting conditions  and for virulence  or high osmolarity (340 mM NaCl) . We also tested if a decrease in pH would affect T6SS expression but could not see a difference when P. phymatum was grown at pH 5.5 or 7, suggesting that pH and acidity do not affect T6SS expression (data not shown). Temperature and growth-phase-dependent expression of T6SSs is reminiscent of regulation of virulence factor production in bacterial pathogens, which can involve various regulatory mechanisms such as the stationary phase sigma factor RpoS, different quorum sensing systems or the secondary messenger c-di-GMP (P. phymatum’s genome contains rpoS (Bphy_0962) and the cepRI quorum sensing system (Bphy_4439-Bphy_4437), as well as several genes involved in c-di-GMP metabolism. Additional work will be required to unravel the molecular basis of the stationary phase induction of T6SS-3.The role of T6SS during the symbiosis between rhizobia and legumes is still poorly understood. Before T6SS were officially discovered in 2006, the hizobium . Two seca plants . The exaion host . In contectively . This phectively . In 2016 species . Burkhol Robbsia  and two inickia) . Interes strains , 14 of wltoides) . P. hospnacearum . Interesnditions . To explpression . While Phe plant . In casehe plant . The fache plant , suggestnditions . Althougnditions  and in P nodules . This su nodules . The invorescens . A BLASTdomallei . More pr T6SS-3; . Althougl models , to our irulence . Analysiirulence . Constitironment . In contmM NaCl) , 2012. Fuillarum . In P. alymyxins . Additiotiveness . Our resc-di-GMP . P. phymP. phymatum. Finally, our study suggests that P. phymatum seemingly activates its T6SS copies depending on the environmental niche it is currently occupying, responding to environmental cues such as carbon sources and temperature. We showed that T6SS-b is occurring in other soil-dwelling bacteria and is activated at temperatures found in the soil and in the rhizosphere, which possibly contains citrate, another inducer of T6SS-b expression. In contrast, T6SS-3 is found mostly in pathogenic Burkholderia strains and is expressed at highest levels at temperatures usually found in animal hosts (37°C) and in an environment containing succinate or aspartate as a carbon source. The identification of T6SS-b and T6SS-3 effector proteins following induction of each T6SS using the corresponding external clues and analyzing the secretome with proteomics represent important follow-up studies that aim at achieving a better molecular understanding of the respective function(s) of these systems that co-occur in genomes of this versatile group of bacteria.Under our experimental settings, we did not identify a role for T6SS-3 in free-living or symbiotic growth conditions. We cannot, however, exclude that in symbiosis with other plants or other host organisms, T6SS-b or T6SS-3 are playing a role or even that the T6SS may have bearing on the exceptional promiscuity of The original contributions presented in the study are included in the article/SH and GP conceived and designed the experiments and wrote the manuscript. SH, YL, and AB performed the experiments. SH, BH, AB, CA, LE, and GP analyzed the data. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Sprague–Dawley (SD) rats (n = 9) were implanted with C6 glioma cells. Tumor growth was monitored every week by multiparameter MRI [including dynamic contrast-enhanced MRI (DCE-MRI)], [18F]FDG, S-[18F]CF3-D-CYS, and [18F]FDOPA PET imaging. Repeated scans of the same rat with the two or three [18F]-labeled radiotracers were investigated. Initial regions of interest were manually delineated on T2WI and set on the same level of PET images, and tumor-to-normal brain uptake ratios (TNRs) were calculated to semiquantitatively assess the tracer accumulation in the tumor. The tumor volume in PET and histopathology was calculated. HE and Ki67 immunohistochemical staining were further performed. The correlations between the uptake of S-[18F]CF3-D-CYS and Ki67 were analyzed. Dynamic S-[18F]CF3-D-CYS PET imaging showed tumor uptake rapidly reached a peak, maintained plateau during 10–30 min after injection, then decreased slowly. Compared with [18F]FDG and [18F]FDOPA PET imaging, S-[18F]CF3-D-CYS PET demonstrated the highest TNRs (P < 0.05). There were no significant differences in the tumor volume measured on S-[18F]CF3-D-CYS PET or HE specimen. Furthermore, our results showed that the uptake of S-[18F]CF3-D-CYS was significantly positively correlated with tumor Ki67, and the poor accumulated S-[18F]CF3-D-CYS was consistent with tumor hemorrhage. There was no significant correlation between the S-[18F]CF3-D-CYS uptakes and the Ktrans values derived from DCE-MRI. In comparison with MRI and histopathology, S-[18F]CF3-D-CYS PET performs well in the diagnosis and evaluation of glioma. S-[18F]CF3-D-CYS PET may serve as a valuable tool in the clinical management of gliomas.Comparing MRI and histopathology, this study aims to comprehensively explore the potential application of  Gliomas are the most common primary intracranial tumor with high mortality and poor prognosis. The high tumor heterogeneity and invasiveness present a considerable obstacle to the treatment of glioma, which was the reason for causing tumor recurrence and treatment resistance . Thus, s18F]FDG is the most commonly used PET tracer in the clinic, and it has been widely used to evaluate a variety of cancers, including gliomas. However, the high uptakes of [18F]FDG in both tumor and normal gray matter bring a strong challenge for the diagnosis of gliomas and tumor boundary delineation. Currently, amino acid (AA) tracers have been used predominantly for glioma imaging and exhibit lower uptake in a normal brain tissue, which is better suitable for the delineation of tumor extent and treatment planning than [18F]FDG (S-[11C]methyl-L-methionine ([11C]-MET) is preferred in clinical use  further limits the extensive clinical application of [11C]-MET  has recently been used for evaluating glioma and demonstrated promising results in predicting low-grade glioma prognosis and in diagnosing recurrent glioma  and similar transport mechanism to [11C]-MET make O-(2-[18F]fluoroethyl)-L-tyrosine  more suitable for clinical use  into consideration, our group has developed a pair of novel 18F-labeled AA PET tracers (18F-trifluoromethylated D- and L-cysteines)  was a promising PET tracer for evaluating gliomas. In addition, S-[18F]CF3-D-CYS is an analog of S-[11C]methyl-L-cysteine, in which its low uptake by the brain tissue has been reported and was able to detect recurrent glioma (S-[18F]CF3-D-CYS PET imaging in the diagnosis of glioma compared to [18F]FDG and [18F]FDOPA PET with reference to MRI and histopathology through orthotropic C6 glioma models.Taking the advantage of steines) . They wet glioma . Hence, This study has complied with the recommendations of the Guidance for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of the People's Republic of China. Our experiments were approved by the Institutional Animal Care and Use Committee (IACUC), Sun Yat-sen University (Permit Number SYSU-IACUC-2019-000057).n = 9) were stereotactically injected with C6 glioma cells for the establishment of orthotropic C6 glioma models. Multiparameter MRI including conventional MRI and DCE-MRI and [18F]FDG PET imaging was performed on the 7th, 14th, and 21st days after the operation. Subsequently, S-[18F]CF3-D-CYS and [18F]FDOPA PET imaging was performed in the succeeding days. Experimental procedures are shown in The male Sprague–Dawley (SD) rats (2 and 95% air at 37°C. Cells were harvested or passaged with trypsinization when they reached over 90% confluence in the logarithmic growth phase.C6 glioma cells were purchased from the Cell Bank of the Chinese Academy of Sciences in Shanghai. Cells were cultivated in culture medium composed of 90% Dulbecco's modified Eagle's medium (GIBCO) and 10% fetal bovine serum (GIBCO) in a cell incubator with a humidified atmosphere of 5% CO6 C6 cells in 10 μL of phosphate-buffered saline (PBS) was injected into the right hemisphere (the right caudate nucleus in more specific terms) of each male SD rat  through a small-animal stereotactic instrument. During the operation, rats were in effective anesthetic condition with 2% pentobarbital (40 mg/kg), and the heads of rats were immobilized. Before cell injections, the skin of the heads was sterilized, and then, skulls were exposed with incisions. Through a burr hole placed 1 mm anteriorly and 3 mm right laterally to the bregma, cells in a 10-μL microsyringe were slowly injected into the white matter at a depth of 5 mm during 10 min. After 5–10 min pace times, the microsyringe was drawn, and the drilled hole was sealed. Furthermore, the wound was sutured, and animals were sent back to a specific pathogen-free laboratory animal room.To establish orthotropic C6 glioma models, about 1 × 10S-[18F]CF3-D-CYS  of 2-mm diameter were drawn over tumor tissues and normal brain tissues  of certain PET/CT images. Furthermore, radioactivity uptake of tissues was presented as mean % ID/g . % ID/g means a percentage of the injected dose per gram of tissue.Two-dimensional ordered-subsets expectation maximum was used during image reconstruction. The 120-min dynamic 2-weighted images CF3-D-CYS reached the maximum accumulation in the tumors at 10 min after injection and with long-term retention; then, it began to slowly decline at 30 min after injection. Until postinjection of 2 h, the uptake of S-[18F]CF3-D-CYS in tumors was still higher than that of the normal brain. The mean uptake ratios of the TNRs are shown in The representative time–activity curves TACs,  showed rS-[18F]CF3-D-CYS were constant from 10 to 30 min p.i., which indicated that the optimal acquisition time for static PET imaging was at 10–30 min after injections. Thus, the frames during the above time period were summed to obtain static images  in the same rat were shown in S-[18F]CF3-D-CYS PET imaging demonstrated the highest mean TNRs  were the highest compared with those of [18F]FDG PET (1.28 ± 0.18) and [18F]FDOPA PET (1.90 ± 0.21)  . Furtherboundary , 4B.S-[18F]CF3-D-CYS PET imaging, MRI, and HE-staining tumor specimen of a representative rat model as an example, which is shown in S-[18F]CF3-D-CYS PET imaging showed high accordance with pathology depicting this C6 glioma with irregular tumor boundary  and the corresponding Ki67 labeling index , indicating that tumor S-[18F]CF3-D-CYS uptake values could reflect the proliferative ability of tumor cells.As shown in S-[18F]CF3-D-CYS PET imaging in reflecting tumor heterogeneity, we performed SWI-MRI examinations to detect the tumor bleeding. Results showed that there was internal hemorrhage for some C6 gliomas, and the bleeding was further confirmed by HE results. Comparing the tumor hemorrhagic site in SWI-MRI and S-[18F]CF3-D-CYS PET images, we found visually low tumor uptakes of S-[18F]CF3-D-CYS in the bleeding region, as shown in the 18F]FDG . DCE-MRI analysis and concentration–time curve results . However, there was an exception that there was no significant difference between the Ktrans values of tumors and that of contralateral normal brain tissues . Thus, the correlations between S-[18F]CF3-D-CYS uptakes and Ktrans values were still debatable, and the tumor uptake of S-[18F]CF3-D-CYS was not necessary to depend on the degree of capillary permeability.All the tumors were vividly enhanced on T results  showed t tissues . Even soS-[18F]CF3-D-CYS) for evaluating glioma in orthotopic C6 glioma models. For S-[18F]CF3-D-CYS PET imaging, there was relatively high tumor uptake and low normal brain uptake. Compared with [18F]FDG and [18F]FDOPA PET imaging, S-[18F]CF3-D-CYS PET imaging had the highest TNRs, which was an advantage for tumor boundary delineation. The tumor volume in S-[18F]CF3-D-CYS PET imaging was close to the pathological size. Besides, we proved that S-[18F]CF3-D-CYS PET imaging could reflect the tumor hemorrhage and was significantly correlated with tumor cell proliferation. Furthermore, our results suggested that the uptake of S-[18F]CF3-D-CYS was independent of capillary permeability.In this study, we successfully further explored and evaluated the application of a new sulfur-containing amino acid PET tracer  and lasted for a long period (for 2 h), which makes the PET acquisition time of S-[18F]CF3-D-CYS flexible. Furthermore, our study showed that the uptake of S-[18F]CF3-D-CYS in the normal brain was relatively low, and it provided a good imaging contrast. Compared with [18F]FDG and [18F]FDOPA PET imaging, S-[18F]CF3-D-CYS PET imaging had the highest mean TNRs; it was possible to clearly define the C6 glioma boundary  and ASC (in the presence of Na+). Thus, we reasonably deduce that LAT-1 can transport S-[18F]CF3-D-CYS in the absence of Na+, and ASCT-2 is likely one of the transporters for uptake of S-[18F]CF3-D-CYS in vitro. These two transporters may play some roles in vivo as well. However, the transport mechanism of S-[18F]CF3-D-CYS is not clear yet. The final and precise conclusions with the tumor uptake mechanism of S-[18F]CF3-D-CYS still need to be further studied. Western blot or mass spectrometry to quantify transporter proteins needs to be further explored.Going even further, the expression of some AA transporters, particularly the system L subtype LAT-1, system ASC subtype ASCT-2, and so on, is increased in malignant lesions , 33. In S-[18F]CF3-D-CYS over common clinical PET agents in imaging glioma. First, as D-isomers of PET enantiopure tracers, S-[18F]CF3-D-CYS, familiar with previous studies such as 3-[18F]fluoro-a-methyl-D-tyrosine (D-[18F]FAMT) and (D)-18F-fluoromethyltyrosine (D-18F-FMT), has some properties compared with their corresponding L-amino acids , whose uptake is facilitated by BBB breakdown , Sun Yat-sen University (Permit Number: SYSU-IACUC-2019-000057).HM, JZ, SL, and GT: design of the study. SL and FW: synthesis of tracers. HM, JZ, and ZZ: animal experiments. DX: image scanning. DN, ZY, and GT: supervision of experiments. HM: original manuscript writing. All authors: discussion and analysis of results, critical feedback, revisions, and approval of the 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.


Secale cereale L.). The ‘Gülzow’ (G)-type cytoplasmic male sterility (CMS) system in hybrid rye breeding exhibits a superior Rf. While having received little scientific attention, one major G-type Rf gene has been identified on 4RL (Rfg1) and two minor genes on 3R (Rfg2) and 6R (Rfg3) chromosomes. Here, we report a comprehensive investigation of the genetics underlying restoration of male fertility in a large G-type CMS breeding system using recent advents in rye genomic resources. This includes: (I) genome-wide association studies (GWAS) on G-type germplasm; (II) GWAS on a biparental mapping population; and (III) an RNA sequence study to investigate the expression of genes residing in Rf-associated regions in G-type rye hybrids. Our findings provide compelling evidence of a novel major G-type non-PPR Rf gene on the 3RL chromosome belonging to the mitochondrial transcription termination factor gene family. We provisionally denote the identified novel Rf gene on 3RL RfNOS1. The discovery made in this study is distinct from known P- and C-type systems in rye as well as recognized CMS systems in barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.). We believe this study constitutes a stepping stone towards understanding the restoration of male fertility in the G-type CMS system and potential resources for addressing the inherent issues of the P-type system.Efficient and stable restoration of male fertility (Rf) is a prerequisite for large-scale hybrid seed production but remains an inherent issue in the predominant fertility control system of rye ( Secale cereale L.) in Northern Europe . I. IRf-like family ,57,58. IExamining the RNA-seq data, none of the eight RFL-PPR genes residing in close proximity to the top associated marker peak were, however, found to be co-expressed at the flowering stage in the G-type hybrids . InsteadRf gene on 1RS, we did not observe any Rf-associated QTLs on 1RS in the mapping population GWAS  the effect of major Rf genes [Rf genes have been identified in the G-type CMS hybrid rye breeding systems on the 3R (Rfg2) and 6R (Rfg3) chromosomes [Rf QTL at 807.1–808.7 Mbp, indicative of a minor Rf gene , 1BS (Rf3), 6AS (Rf9), 6BS , 6D (Rf5), and 7D (Rf2) chromosomes [Hordeum vulgare L.), two major Rf genes have been identified on 6HS  [Brachypodium distachyon L., rice (Oryza sativa L.), sorghum (Sorghum bicolore L.), and barley. Rye 4RL, a region housing three major P-type and one C-type Rf genes, was found to be syntenic to barley 6HS [Rfp1 on 4RL exhibits an ortholog on wheat 6DS and barley 6H. In a subsequent study, Hackauf et al. [Rfp3 on 4RL likely maps to an orthologous segment housing Rf6 on wheat 6BS and Rfm1 on barley 6HS [Rf genes pivotal to the fertilization control systems across rye (P- and C-type), barley, and wheat [Rf gene, RfNOS1, on 3RL in the G-type CMS system in rye, with no known Rf genes on orthologous chromosome segments in other Triticeae species.To our knowledge, no omosomes ,61,62,631, Rfm3) ,64. Intr1, Rfm3)  discoverrley 6HS ,65. Thesrley 6HS , who repf et al. , furthernd wheat . TogetheRf genes have been assigned to the PPR superfamily, denoted as Rf-like PPR genes or RFL-PPRs. In domesticated Poaceae species, this includes wheat Rf1 and Rf3 [Rfm1 [Rf1 [Rf5 [Rf4, Rf5, and Rf6 [Rf gene RfNOS1 in the ‘Lo7’ reference genome revealed no RFL-PPR genes. Instead, the region harbored five PLS-class PPR genes either misclassified, not resembling known PLS-type RFL-PPR genes, or pointing towards a non-PPR Rf gene on 3RL  [Arabidopsis, KATANIN is required for fertility, embryo development, and seed production [A growing body of in rice; ), acyl-cin rice; ), aldehyin rice; ), bHLH tin rice; ), and Rfaris L.) . The regoduction . Genes ioduction ,73,74, Boduction , and basoduction  were likRfp1 and Rfp3 on 4RL to mTERF genes. A similar observation was made by Bernhard et al. [Rfm3 on barley 6HS, syntenic to rye 4RL. Pan et al. [Rf gene on 3RL. We denote the gene as RfNOS1, belonging to the expanding class of non-PPR Rf genes.Intriguingly, the region was found to harbor a single mTERF gene located at 731 Mbp, coinciding with the top-associated peak in the 600K case control GWAS . In rye,d et al. , who iden et al.  successiRf gene on 3RL in either R, S, or C-type CMS system, such a unilateral grouping as V-type seems premature. From our observations, the G-type CMS system distinguishes itself from the other CMS systems by a less pivotal role of Rf genes on 4RL.On the basis of male fertility restoration requirements and genetic similarity of sterilizing cytoplasms, G-, C-, and R-type CMS systems have been proposed to belong to the larger Vavilovii (V) type ,77,78. ISecale cereale L.) elite breeding lines were selected for this study, comprising 242 restorers, 116 non-restorer germplasm (NRG), and 7 cytoplasmic male sterile (CMS) lines. The CMS male sterility is based on the ‘Gülzow’ (G)-type cytoplasm originating from the population of the rye variety ‘Schlägler alt’ [TM microplate spectrophotometer  and evidence of fragmentation by size visualization on a 1.2% agarose gel.In total, 365 Nordic Seed Germany GmbH inbred hybrid rye  were sown in pots containing a course-grain sphagnum substrate at Nordic Seed Germany GmbH greenhouse facilities. The seedlings were cultivated under a 16 h light regime with night temperatures of 14–16 °C and day temperatures of 18–24 °C. Seven days after sowing, at the 2-leaf stage, seedlings were set to vernalize in a climate chamber under 16 h of light at 8 °C for a week and then 3 °C for the following seven weeks. After vernalization, the pots were transferred to the greenhouse. Prior to anther-protrusion, cellophane bags were put on the spikes to prevent cross-fertilization. At maturity, seeds of a single F1 plant were harvested and the procedure was repeated to generate an F2 biparental mapping population. At four timepoints, a total of 181 F2 plants were phenotyped for pollen production using a customized visual 1–9 scale . The plants were, furthermore, scored for number of spikes per plant, total seed number, seeds per spike, total grain weight, and thousand kernel weight in order to obtain a comprehensive phenotypic dataset on the inheritance of male fertility restoration in the population. The segregation ratio of infertile and fertile F2 plants was tested for goodness of fit to the expected Mendelian ratio at the scenario of one, two, and three major restorations of male fertility (Rf) genes using an χ2 test [2 plant was considered ‘sterile’ if it either yielded less than 20 seeds or scored ≤ 2 in pollen production.To investigate the inheritance of male fertility restoration in the G-type CMS-based Nordic Seed breeding system, a biparental mapping population was developed. The population was phenotyped for restoration of male fertility, including traits associated with restoration. Seeds of the hybrid cv. Stannos  R package with LD set as the coefficient of determination (r2) [All rye lines included in this investigation were genotyped using a custom Illumina Infinium 15Kwheat  and 5KRyeat [Rye ,83 singlg et al. . The map top hit ,84. The ion (r2) .Genetic analysis of SNP marker data was performed in R studio  interface in R statistical software (v. 3.6.3) by application of various predesigned packages ,87. PrioRf-associated regions was made by a genome-wide association study (GWAS) using the genomic association and prediction integration tool (GAPIT) (v.3) package in R [2 population, and a binary case control for the entire population relative to their population origin using the 20K SNP array and 600K high-density SNP array, respectively. A standard Bonferroni-corrected threshold of α = 0.05 was used as the significance thresholdThe discovery of age in R . PhenotyRf-associated genes, pentatricopeptide repeat proteins (PPR), Rf-like PPR, and mitochondrial transcription termination factors (mTERF), residing in regions associated with Rf were identified in the ‘Lo7’ reference genome [https://www.ncbi.nlm.nih.gov/bioproject/PRJNA612415, accessed on 12 March 2021). Coding and protein sequences of the Rf-associated gene panel were extracted from the ‘Lo7’ reference genome repository. The de novo assembled transcriptome assemblies were likewise translated to coding protein sequences with TransDecoder . The NCBI blastx and blastp  functions were used to blast coding and protein sequences of Rf-associated genes in the panel towards the generated protein transcriptome database at a significance threshold of the e-value at 10−5 to ascertain genes expressed in the two G-type hybrid cv. at flowering stage [Annotated major e genome . To ascee genome  and raw ng stage .https://inkscape.org/, accessed on 7 May 2021).Graphs and figures were outputted from R in .svg format and manually curated using Inkscape (v. 1.1) program (Rf gene on 3RL with no known orthologues in either barley or wheat. Gene mining of the Rf-associated region on 3RL led to the identification of an mTERF gene co-expressed in two G-type hybrids as a candidate gene for restoration. We propose to denote the novel G-type Rf gene as RfNOS1. Conclusively, our investigation provides a novel insight into the genetics of male fertility restoration in a G-type CMS system and its differentiation to rye P- and C-types in addition to known CMS systems in barley and wheat.In this study, we exploited recent advents in rye genomic resources to dissect the genetics underlying restoration of male fertility in a G-type CMS system. Our findings provide compelling evidence of a novel major G-type


Here, by importing cobalt single clusters as strong electron-donating promoter into the catalyst, the rate-determining step of ammonia synthesis is altered to the subsequent proton addition so that the barrier of N2 dissociation can be successfully overcome. As revealed by density functional theory calculations, the N2 dissociation becomes exothermic over the cobalt single cluster upon the strong electron backdonation from metal to the N2 antibonding orbitals. The energy barrier of the positively shifted rate-determining step is also greatly reduced. At the same time, advanced sampling molecular dynamics simulations indicate a barrier-less process of the N2 approaching the active sites that greatly facilitates the mass transfer. With suitable thermodynamic and dynamic property, a high ammonia yield rate of 76.2 μg h–1 mgActivation of high-energy triple-bonds of N The most significant bottleneck of ambient ammonia synthesis, namely nitrogen activation, has been successfully overcome by altering the rate-determining step over cobalt single clusters-contained electrocatalyst, and superior nitrogen reduction performance was achieved. Unfortunately, only very few catalysts reported to date can efficiently reduce the nitrogen activation barrier, leaving the ammonia production rate and Faradaic efficiency in low level [The electrocatalytic Nnditions . Theoretform NH3 . Yet, sting step ,19. A cag to, N2 . The bacg effect , and furow level . Hence, 2 cleavage to proton addition  calculations reveal that N2 activation is transferred into a strongHerein, we successfully demonstrate that deliberately introducing cobalt single clusters as electron-donating promoter in nitrogen-doped carbon alters the rate-determining step of ammonia synthesis from Nion Fig. a. An excx formation in the whole nitrogen fixation process. Also, the approaching process of N2 molecules towards the single cluster sites is confirmed to be barrier-less by molecular dynamics (MD) simulations, which greatly favors the whole nitrogen reduction process. Altering the rate-determining step of nitrogen reduction effectively leads to a desirable NRR performance, and thus provides a powerful guidance for future design of catalysts.thermodynamically exothermic process on cobalt single clusters, so that it is no longer the rate-determining step of ammonia synthesis. Instead, only small energy barriers exist upon NHSC-N-C) were fabricated and carefully characterized. The transmission electron microscopy (TEM) image  and dark-field TEM image  analysis. To highlight the specific role of cobalt single clusters, counterparts with cobalt nanoparticles (CoNP-N-C) and without metal (N-C) were also synthesized by replacing the Co2+/Zn2+ mixture with single Co2+ and Zn2+, respectively, under otherwise identical conditions . As shown in the X-ray powder diffraction (XRD) patterns , CoNP-N-C exhibits distinct metallic cobalt diffraction, whereas the cobalt single clusters in CoSC-N-C exist as amorphous phase. The increased content of Co accounts for the improved graphitization degree, as evaluated by the Raman spectra .The cobalt single clusters dispersed in nitrogen-doped carbon  spectrum of the CoSC-N-C . The coexistence of four different N species in different samples, namely pyridinic-N, pyrrolic-N, graphitic-N and N-oxides, was confirmed by the high-resolution N 1s spectra  [SC-N-C compared with CoNP-N-C [SC-N-C. In addition, CoSC-N-C possesses a large surface area of 287.1 m2 g−1, as determined by the Brunauer-Emmett-Teller (BET) method .To confirm the chemical state of Co species in different samples, X-ray absorption fine structure (XAFS) measurements were conducted with Co foil as reference. The Co K-edge X-ray absorption near-edge structure (XANES) shows that the absorption edge of CoN-C Fig. d. The FoN-C Fig. e shows t Fig. 8) ,27. The CoNP-N-C ,29, indi2 clearly affirms the contribution from the nitrogen reduction. Without cobalt sites, N-C exhibits the weakest NRR and hydrogen evolution reaction (HER) activity. While, compared with CoNP-N-C, the bigger current density gap between Ar and N2 and lower HER current density of CoSC-N-C indicate that the introduction of cobalt single clusters leads to much more improved NRR activty and selectivity than those of the cobalt nanoparticles. Then, a quantified study of the NRR ability for different samples was carried out via chronoamperometry measurement using an H-shape electrochemical cell [3 and NOx. Possible reduction products including NH3 and N2H4 were both tested , whereas only NH3 was detected in this work . Before the NRR test, several control experiments were first carried out to ensure that no contamination was present in the feeding gas or the equipment, and the carbon paper (CP) as current collector of the cathode did not have NRR activity, so that NH3 could only be produced by N2 reduction in the presence of catalyst , exhibits the highest NH3 yield rate of 76.2 μg h–1 mgNP-N-C , which is more than one order of magnitude higher than the counterparts. Detailed chronoamperometry responses are shown in Supplementary Fig. 18. Also, the electrochemical active surface area (EASA) of each sample was determined by double-layer capacitance . Expectedly, the surface-area-normalized ammonia production rate of CoSC-N-C shows an obvious advantage over other samples . The NRR activity and selectivity of CoSC-N-C remarkably stands at the top level of reported catalysts under mild conditions (Supplementary Table 1), and is thus of great significance in energy utilization. Based on the nitrogen temperature-programmed desorption (N2-TPD) spectra , CoSC-N-C exhibits the strongest N2 adsorption ability, confirming that cobalt single clusters are superior nitrogen-adsorption active sites compared with cobalt nanoparticles or nitrogen-doped carbon [SC-N-C as the applied potential becomes more negative than −0.2 V vs. RHE is mainly due to the increased HER [SC-N-C shows a much smaller proportion of the competing HER compared with CoNP-N-C and N-C, and is more inclined to proceed NRR . Based on the above results, CoSC-N-C has a prominent advantage in nitrogen reduction, especially at −0.2 V vs. RHE  curves were first measured . The current density difference between Ar and Ncal cell . Here, tcal cell . The nitd carbon . The graased HER , as eval3 is helpful in getting an in-depth understanding of the catalyzing mechanism. Thus, isotope-labeling experiments were systematically conducted [15N2 gas was also sufficiently purified before use. As shown in the ultraviolet-visible (UV–vis) absorption spectra  spectra  representing 15NH4+ is detected in the spectra instead of the triplet signal (∼52 Hz) of 14NH4+. When Ar was used for electrolysis, no NH3 signal was detected in the NMR spectrum, indicating the obtained ammonia is totally from the N2 electroreduction process. For accuracy of the performance data, NMR method was also employed for ammonia quantification , manifesting its broad prospect for practical applications. Simultaneously, TEM image, corresponding element maps and Raman spectra  exhibit no variation in the morphology and structural properties of CoSC-N-C after the NRR process. Its chemical state is also well maintained as confirmed by the high-resolution XPS spectra analyses , demonstrating that the CoSC-N-C is robust enough for long-term NRR electrocatalysis.Stability is another vital parameter for an electrocatalyst. Thus, the durability of CoSC-N-C catalyst. The thermodynamic process of different models was first studied by DFT calculations. As confirmed by catalyst characterizations, the cobalt in CoSC-N-C mainly exists as single clusters. Accordingly, several possible models of cobalt single cluster with different numbers of cobalt atoms on nitrogen-doped carbon  were systematically proposed, and pure nitrogen-doped carbon (N4/C) was also calculated for comparison . For N4/C, the nitrogen adsorption is a strong endothermic process with a step-by-step uphill trend of Gibbs free energy . The introduction of cobalt single clusters is able to turn the nitrogen adsorption into exothermic process without energy barriers, suggesting that the cobalt single clusters are superior nitrogen-adsorption active sites. Notably, all of the structures with cobalt single clusters are able to alter the rate-determining step to the subsequent nitrogen hydrogenation with relatively small energy barriers . Taking Co4-N4/C for example, as clearly shown in Fig. 2 adsorption, the preferred NRR approach of Co4-N4/C is verified to be the associative alternating pathway instead of the associative distal pathway . When chemically adsorbed on Co cluster, N2 is spontaneously activated and experiences a significant weakening of the N≡N bond due to the strong electron backdonation from the metal to the N2 antibonding orbitals, and the N2 dissociation becomes an exothermic process over the cobalt single cluster. In addition, the energy released from the N2 adsorption step greatly benefits the following N2 cleavage, according to the ‘hot atom’ mechanism [2 activation to the subsequent hydrogenation with only a small energy barrier of 0.85 eV. In great contrast, without the Co single cluster, the N4/C model not only suffers a severely endothermic N2 adsorption process, but also possesses high NRR rate-limiting barriers of 2.04 eV and 1.84 eV for the alternating and distal pathways, respectively, indicating a weak activity towards NRR  with a step-by-step downhill trend of the Gibbs free energy, whereas the H chemisorption suffers an extremely high energy barrier of 2.53 eV due to the water dissociation process. Even if *H is adsorbed, its desorption to form H2 is still an endothermic reaction , which is also beneficial to promoting the NRR Faradaic efficiency.Computational studies on both thermodynamics and dynamics were carried out to investigate the mechanism of ammonia synthesis over the Coechanism ,38. Thus2 approaching process towards Co4-N4/C was then explored by advanced sampling MD simulations. The system was set up by randomly placing 6 K+, 6 OH− and 3000 water molecules in the simulation box, with a Co4-N4/C model fixed perpendicular to the z-axis at the center of the simulation system. The N2 molecule initially located at 1.5 nm above the geometric center of the upper three Co atoms. Then, the N2 molecule was pulled towards the Co4-N4/C along the z-axis at a rate of 0.01 nm ps–1 under a harmonic force constant to generate configurations for umbrella-sampling (Fig. 2 molecule is nearly barrier-less with only a small energy hill at approximately 0.52 nm. This clearly indicates that the cobalt single cluster as active site is accessible to the N2 molecule, which can greatly promote the following N2 chemisorption, and is thus contributory to the entire NRR process. The above computational conclusions well explain the experimentalFrom a dynamic point of view, the N2 fixation.results, and further highlight the contribution of the cobalt single cluster as an important promoter for ambient N3 synthesis by deliberate introduction of cobalt single clusters as electron-donating promoter in nitrogen-doped carbon, and achieved outstanding ammonia yield rate of 76.2 μg h–1 mgSC-N-C is steady enough for long-term NRR electrocatalysis with negligible decay of the amazingly high Faradaic efficiency through consecutive electrolysis recycle. The 15N isotopic labeling experiments and control experiments are combined to confirm that the N source of N2-to-NH3 conversion is completely from the feeding gas instead of any activated N species in the catalyst. First-principles simulations demonstrate a strong exothermic process of N2 chemisorption on the cobalt single cluster site, which greatly promotes the following N2 dissociation. The N2 activation, therefore, becomes exothermic, and the rate-determining step has been successfully altered to the subsequent nitrogen hydrogenation. A smooth N2 approaching process with almost no energy hill towards the catalyst paves the way for the N2 mass transfer, as confirmed by the MD simulations. Our work overcomes the obstacle of ambient ammonia synthesis, and contributes to the guiding of future catalyst design for sustainable NRR systems.In summary, we have successfully altered the rate-determining step of ambient NHnwaa136_Supplemental_FileClick here for additional data file.


Plant cells perceive and adapt to an ever-changing environment by modifying their plasma membrane (PM) proteome. Whereas secretion deposits new integral membrane proteins, internalization by endocytosis removes membrane proteins and associated ligands, largely with the aid of adaptor protein (AP) complexes and the scaffolding molecule clathrin. Two AP complexes function in clathrin-mediated endocytosis at the PM in plant cells, the heterotetrameric AP-2 complex and the hetero-octameric TPLATE complex (TPC). Whereas single subunit mutants in AP-2 develop into viable plants, genetic mutation of a single TPC subunit causes fully penetrant male sterility and silencing single subunits leads to seedling lethality. To address TPC function in somatic root cells, while minimizing indirect effects on plant growth, we employed nanobody-dependent delocalization of a functional, GFP-tagged TPC subunit, TML, in its respective homozygous genetic mutant background. In order to decrease the amount of functional TPC at the PM, we targeted our nanobody construct to the mitochondria and fused it to TagBFP2 to visualize it independently of its bait. We furthermore limited the effect of our delocalization to those tissues that are easily accessible for live-cell imaging by expressing it from the PIN2 promoter, which is active in root epidermal and cortex cells. With this approach, we successfully delocalized TML from the PM. Moreover, we also show co-recruitment of TML-GFP and AP2A1-TagRFP to the mitochondria, suggesting that our approach delocalized complexes, rather than individual adaptor complex subunits. In line with the specific expression domain, we only observed minor effects on root growth, yet realized a clear reduction of endocytic flux in epidermal root cells. Nanobody-dependent delocalization in plants, here exemplified using a TPC subunit, has the potential to be widely applicable to achieve specific loss-of-function analysis of otherwise lethal mutants. Cells are delineated by their plasma membrane (PM). The PM houses a plethora of proteins ranging from receptors and ion channels to structural membrane proteins. Many of these PM proteins, commonly termed cargo, are responsible for cellular communication with the outside world. In eukaryotes, endocytosis is the cellular process where cargoes, associated ligands as well as lipids are internalized from the PM. Endocytosis thereby provides a way to regulate the content and consequently modulate protein activity at the PM. A predominant and well-studied form of endocytosis is clathrin-mediated endocytosis CME; . CME refDictyostelium discoideum contains a similar complex, named TSET. TSET, however, is a hexameric complex in contrast to TPC in Arabidopsis thaliana, which has two additional subunits. Also contrary to TPC, TSET is dispensable in D. discoideum  and the hetero-octameric TPLATE complex (TPC) facilitate CME in plants . In contscoideum . The prescoideum .ap2 mutants show reduced internalization of the styryl dye N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl) Hexatrienyl) Pyridinium Dibromide (FM4-64), which can be seen as proxy to a difference in cargo uptake , and the cellulose synthase subunit CESA6 , have found their way into a wide variety of applications in biological fields. Nanobodies are similar to antibodies (Ab) in the sense that they can bind epitopes with high affinity in a highly selective manner . Their aDrosophila melanogaster. DeGradFP links an anti-GFP nanobody to an F-box protein, thereby targeting it for ubiquitin-dependent degradation (Arabidopsis flowering meristem (Arabidopsis in transgenic tobacco plants (Arabidopsis seedlings to lock down vacuolar sorting receptors (VSRs) in cellular compartments upstream of trans-Golgi network/early endosomes, allowing to determine their retrograde trafficking pathway  mutant background and delocalized it to the mitochondria using a nanobody directed against eGFP.Here, we explore, similar to what has been done in animal cells , the effGateway entry clones pDONR221-TagBFP2, pDONR221-MITOTagBFP2 and pDONRP2RP3-GFPNb were generated according to the manufacturer’s instructions (ThermoFisher Scientific BP clonase). pDONR221-TagBFP2 was amplified from pSN.5 mTagBFP2  with priAttB1-GGGGACAAGTTTGTACAAAAAAGCAGGCTATGTCATCTAAGGGTGAAGAGC TTATC AAAGAGAAT and AttB2-GGGGACCACTTTGTACAAGAAAGCTGGGTCACCTCCGCCACCTCCACCTCCCAGTCCTGCGTA.pDONR221-MITOTagBFP2 was generated from pDONR221-TagBFP2 by including the import signal of the yeast mitochondrial outer membrane protein Tom70p as described before . The folAttB1-GGGGACAAGTTTGTACAAAAAAGCAGGCTCAATGAAGAGCTTCATTACAAGGAACAAGACAGCCATTTTGGC AACCGTTGCTGCTACAGGTACTGCCATCGGTGCCTACTATTATTACAACCAATTGCAACAGGATCCACCGGTCGCCACCATGTCATCTAAGGGTGAAGAGCTT and AttB2-GGGGACCACTTTGTACAAGAAAGCTGGGTACGCTAAGTCTTCCTCTGAAATCAA.pDONRP2RP3-GFPNb was generated from an anti-GFP Nanobody construct  with priThe entry clones of the PIN2 promoter pDONRP4P1R_PIN2prom  or 35 s Nicotiana benthamiana plants were grown in a greenhouse under long-day conditions  in soil (Saniflo Osmocote pro NPK: 16 − 11 − 10+ magnesium and trace elements). Transient expression was performed by leaf infiltration according to (rding to . The abatml-1(−/−) (At5g57460) mutant lines described previously (tml-1(−/−) mutant line expressing also RPS5Aprom::AP2A1-TagRFP (via microscopy and best lines were selected on the basis of both PIN2prom::MITOTagBFP2-GFPNb and RPS5Aprom::AP2A1-TagRFP expression. For both Col-0 and tml-1(−/−) backgrounds, two independent lines (-Nb1 and -Nb2) were generated. Namely, Col-Nb1, Col-Nb2, TML-Nb1, and TML-Nb2. In order to synchronize the age and overall fitness of the seeds, all lines used in the root growth and carbon starvation study, including the Col-0 and tml-1(−/−) lines, were propagated together and collected at the same time.Plants expressing pB7m34GW_PIN2prom::MITOTagBFP2-GFPNb were generated by floral dip . Construeviously . Primary1-TagRFP . Primaryp-values were adjusted using the Bonferroni method.Four to five days old seedlings of TML, TML-Nb1, and TML-Nb2 were grown on ½ strength MS medium without sucrose and 0.6% Gelrite . Acquired pictures were analyzed in Fiji/ImageJ . In eachArabidopsis seedlings were grown on ½ strength MS medium without sucrose and 0.6% Gelrite . Seeds of all lines were equally placed on the plates . Plates were left for the stratification for 48 h at 4°C, and then placed at 21°C in continuous light. Two days after transfer to the light, seeds which did not germinate were marked and excluded in the further analysis. For root growth analysis, seedlings were grown in continuous light and 2 days after germination the root growth of every seedling was marked for 7 days, daily. For carbon starvation, seedlings were grown for 5 days, including the germination period, in continuous light after which the root growth of every seedling was marked. Subsequently, the plates were covered with aluminum foil and left for 7 days in dark after which root growth was marked again. Root growth and carbon starvation assays measurements were carried out with Fiji/ImageJ . Fixed effects in the model were Line, Day, and the interaction term. An unstructured covariance structure was estimated to model the correlations between measurements done on the same plant. The degrees of freedom of the fixed effects were approximated with the Kenward-Rogers method. The hypotheses of interests were the differences between Col-0 and its respective nanobody lines, between TML and its respective nanobody lines and between the nanobody lines with the same background. These hypotheses were tested using the plm procedure. p-values were adjusted for multiple testing using the maxT procedure as implemented in the plm procedure. For the carbon starvation assay, statistical differences in root growth between the lines were analyzed with pairwise Wilcoxon tests. p-values were adjusted using the Bonferroni method.i/ImageJ . Seedlinvia interquartile range  in a single step. The normality assumption of the measurements was verified with the Shapiro-Wilk normality test. Due to violation of the normality assumption, statistical differences in intensity values between the lines were analyzed with pairwise Wilcoxon tests. p-values were adjusted using the Bonferroni method. All Shapiro-Wilk normality tests and Wilcoxon tests were performed using the R statistical software. . Roots were stained with 2 μM FM4-64 by incubating the seedlings in FM-containing ½ strength MS medium without sucrose for 30 min. Treatment was followed by microscopy. Acquired pictures were analyzed in Fiji/ImageJ . PM and Nicotiana benthamiana infiltration assay imaging  mutant expressing TMLprom::TML-GFP (tml-1(−/−) Arabidopsis roots, TML-GFP is recruited predominantly at the PM  and expressing PIN2prom::MITOTagBFP2-GFPNb with the homozygous complemented tml-1(−/−) line, expressing TML-GFP as well as one of the large AP-2 subunits, AP2A1, fused to TagRFP  mutant background: TML-Nb1 (−/−) and TML-Nb2 (−/−). At the seedling level, we did not observe any major adverse developmental effects , TML-Nb1 (−/−), and TML-Nb2 (−/−). We observed a slight decrease in endocytic flux when comparing wild type seedlings with the complemented tml-1(−/−) line and a strong reduction in endocytic flux between the complemented mutant and both complemented mutant lines where TML was partially delocalized  mutant line expressing TML-GFP  mutants, newly synthesized TML-GFP would be broken down immediately, preventing to achieve functional levels of TPC at the PM. Stronger or inducible promotors and/or the use of a different targeting location might also increase the delocalization capacity. To avoid lethality due to ubiquitous sequestration, engineered anti-GFP nanobodies, whose affinity can be controlled by small molecules, could also be used (Recent results suggest that plant cells very likely contain a feedback loop controlling TPC expression, as carbon starved plants contained roughly the same amount of full-length TPLATE-GFP, next to an extensive amount of TPLATE-GFP degradation products . In case be used .via TML resulted in the co-delocalization of one of the large subunits of AP-2, indicating that we likely are not only targeting TPC, but also AP-2 function. Whether a complementary approach, by delocalizing AP-2, using AP2S or AP2M in their respective complemented mutant backgrounds, would also delocalize TPC is something that would be worth trying. Furthermore, as AP2S and AP2M subunits are still recruited in ap2m and ap2s single mutant backgrounds (C. elegans (ap2 mutants and a similar approach as performed here might also provide tools to inactivate AP-2 as a whole, which can be highly complementary to working with the single subunit mutants.Untangling the function of TPC and AP-2 in CME at the PM requires tools that allow interfering specifically with the functionality of both complexes. Our nanobody-dependent approach targeting TPC kgrounds , AP-2 in elegans . Single In conclusion, the data presented here is a first step toward the development of specific tools, which are required to help us understand the functions of AP-2 and TPC. In the long-term, this will generate insight into endocytosis at the mechanistic level and this will bring us closer to being able to modulate CME-dependent processes, and thereby modulating plant development, nutrient uptake as well as defense responses to our benefit.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.JW, ADM, EM, and PG designed and performed experiments. DVD designed experiments and wrote the initial draft together with ADM and JW. VS performed root growth assay statistical analysis. All authors contributed to the article and approved the submitted version.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.


The outbreak of coronavirus disease 2019 (COVID-19), by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has quickly developed into a worldwide pandemic. Mutations in the SARS-CoV-2 genome may affect various aspects of the disease including fatality ratio. In this study, 553,518 SARS-CoV-2 genome sequences isolated from patients from continents for the period 1 December 2020 to 15 March 2021 were comprehensively analyzed and a total of 82 mutations were identified concerning the reference sequence. In addition, associations between the mutations and the case fatality ratio (CFR), cases per million and deaths per million, were examined. The mutations having the highest frequencies among different continents were Spike_D614G and NSP12_P323L. Among the identified mutations, NSP2_T153M, NSP14_I42V and Spike_L18F mutations showed a positive correlation to CFR. While the NSP13_Y541C, NSP3_T73I and NSP3_Q180H mutations demonstrated a negative correlation to CFR. The Spike_D614G and NSP12_P323L mutations showed a positive correlation to deaths per million. The NSP3_T1198K, NS8_L84S and NSP12_A97V mutations showed a significant negative correlation to deaths per million. The NSP12_P323L and Spike_D614G mutations showed a positive correlation to the number of cases per million. In contrast, NS8_L84S and NSP12_A97V mutations showed a negative correlation to the number of cases per million. In addition, among the identified clades, none showed a significant correlation to CFR. The G, GR, GV, S clades showed a significant positive correlation to deaths per million. The GR and S clades showed a positive correlation to number of cases per million. The clades having the highest frequencies among continents were G, followed by GH and GR. These findings should be taken into consideration during epidemiological surveys of the virus and vaccine development. The novel betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for coronavirus disease 2019 (COVID-19), was first described in Wuhan, China in December 2019 . SubsequSARS-CoV-2 genetic mutations may allow the virus to adapt to a new host cell or alter virus pathogenesis . TherefoIn this study, we evaluated 553,518 SARS-CoV-2 genome sequences isolated from different continents and explored their associations with CFR, cases per million and deaths per million.https://www.gisaid.org/ (accessed on 1 December 2020)).Full-length viral nucleotide sequence of the reference SARS-CoV-2 Wuhan-Hu-1 strain (Accession NC_045512) [https://www.gisaid.org/epiflu-applications/covsurvermutations-app/ (accessed on 1 December 2020)) [Only high-coverage SARS-CoV-2 sequences were downloaded from GISAID EpiCoVTM database . Each ofr 2020)) ,13. Aftehttps://www.gisaid.org/references/statements-clarifications/clade-and-lineage-nomenclature-aids-in-genomic-epidemiology-of-active-hcov-19-viruses/ (accessed on 1 December 2020)).Genomic clades were inferred by GISAID database and defined according to its nomenclature system at the time of data collection outlined in (https://www.wfiveorldometers.info/coronavirus/ (accessed on 1 December 2020)). The CFR was calculated by dividing total deaths by total confirmed cases in each country/region.The number of deaths and confirmed cases related to COVID-19 was collected from the Worldometer (In comparison with the reference sequence of SARS-CoV-2 Wuhan-Hu-1 strain (Accession NC_045512.2), phylogenetic analysis was performed for the complete coding region of all isolates using Nextstrain software .The study was carried out following the Helsinki Declaration. Ethical approval was obtained from the Institutional Review Board at the American University of Madaba (Ref. H20010).p) were used to identify the correlation between mutation frequencies with CFR, cases per million and deaths per million. A p-value of less than 0.05 was considered to be statistically significant.The data were analyzed using the Social Sciences Statistical Package version 23 . Hierarchical cluster analysis was used to identify mutant clusters among the countries/regions. Spearman’s correlation (r) values and two-tailed significance  values between the SARS-CoV-2 clades and each of the CFR, cases per million and deaths per million. Among the identified clades, none showed a significant correlation to CFR. The G, GR, GV, S clades showed a significant positive correlation to deaths per million. The GR and S clades showed a significant positive correlation to number of cases per million.p) values between the SARS-CoV-2 mutations and each of the CFR, cases per million and deaths per million. Among the identified mutations, NSP2_T153M, NSP14_I42V and Spike_L18F mutations showed a positive correlation to CFR , 20F, 20G, 20H/501Y.V2, 20I/501Y.V1 and 20J/501Y.V2) by Nextstrain nomenclature, are also known as clades G, GR and GH by GISAID nomenclature .The current COVID-19 outbreak has spread rapidly worldwide. Most COVID-19 infected patients suffering from COVID-19 are either asymptomatic or experience mild symptoms. Around 15% of all COVID-19 patients progress to severe pneumonia and approximately 5% of COVID-19 patients have acute respiratory disorder syndrome, septic shock and multiple organ failure . The CFRThis study explored and compared mutation profiles of SARS-CoV-2 isolates from different continents. We identified differences among continents relating to COVID-19 CFR, cases per million and deaths per million. The differences may be attributed to differences in age distributions ,18, blooLike other RNA viruses, genetic variation in SARS-CoV-2 is important for fitness, survival and pathogenesis. Spontaneous mutations and recombination are two main sources of genetic variation for SARS-CoV-2 .−4 nucleotides/genome per year [Based on data obtained from a public database GISAID for the period 1 December 2020 to 15 March 2021, the mutation rate of the SARS-CoV-2 virus is about 8 × 10per year , which iper year ,28,29. Aper year . The ideper year .The mutation is one of the most effective evolutionary mechanisms in RNA viruses . SARS-CoOur study indicated a significant correlation between the number of both the number of cases per million and the number of deaths per million, with the Spike_D614G mutation, which is a mutation outside the receptor-binding domain (RBD). In line with previous studies, this study revealed that the Spike_D614G mutation showed a positive correlation with viral infectivity and enhanced transmissibility ,40,41.The genome of SARS-CoV-2 is about 30 kb and codes for 14 open frames (ORFs). 5′-ORF-1a/1b forms 67% of the genome and encodes polyproteins 1a (pp1a) and polyproteins 1 ab (pp1ab), which are then processed into sixteen non-structural proteins (nsp1-16) . Other nThe trimetric viral spike (S) glycoprotein is the main determinant of coronaviruses’ host specificity and pathogenesis. The S2 domain activates the viral and host cell membranes’ fusion to encourage viral penetration and uncoating ,46. In oA recent report have shown that the crystal structure of the SARS–CoV-2 nucleocapsid (N) protein is identical to that of the known coronavirus N proteins, but their surface electrostatic potential properties were distinct . Sheik eThe phylogenetic analysis indicated that several virus clades were deployed in different continents and it was observed that clades 20 became dominant. Clades 20 included 20A, 20B, 20C, 20E(EU1), 20F, 20G, 20H/501Y.V2, 20I/501Y.V1 and 20J/501Y.V2 by Nextstrain nomenclature, which is also known as clades G, GR and GH by GISAID nomenclature ,52,53. TThe mutations having the highest frequencies among continents were Spike_D614G and NSP12_P323L. Among the identified mutations, NSP2_T153M, NSP14_I42V and Spike_L18F mutations showed positive correlation to CFR, while NSP13_Y541C, NSP3_T73I and NSP3_Q180H showed a negative correlation. The Spike_D614G and NSP12_P323L mutations showed positive correlation to deaths per million, while NSP3_T1198K, NS8_L84S and NSP12_A97V mutations showed negative correlation. NSP12_P323L and Spike_D614G mutations showed positive correlation to number of cases per million, while NS8_L84S and NSP12_A97V mutations showed negative correlation. In addition, among the identified clades, none showed a significant correlation to CFR. The G, GR, GV, S clades showed a significant positive correlation to deaths per million. The GR and S clades showed a positive correlation to number of cases per million. The clades having the highest frequencies among continents were G, followed by GH and GR. Further confirmation and biological significance studies are needed to verify association of identified mutations and clades with a CFR, number of deaths per million and number of cases per millionCase fatality ratios, number of deaths per million and number of cases per million may be affected by several factors including age distribution differences, blood groups, gender, virus genomic types and ethnic backgrounds; many of these factors still require confirmation.


X-ray diffraction and thermogravimetric analyses confirmed that the consumption of brucite and the formation of M-S-H were quicker with a 50 °C curing. Energy-dispersive X-ray spectroscopy and microtomography showed that colloidal silica enabled a better distribution of the particles than silica fume. Microstructural characterizations were conducted under the protocol with colloidal silica and 50 °C thermal curing. Porosity investigations allowed to describe the M-S-H pastes as highly porous materials with a low content of micropores in comparison with mesopores. The type of mixing influenced the mesopore size distribution.Concretes can be exposed to a magnesium attack in several environments leading to the formation of magnesium silicate hydrates (M-S-H) and brucite (MH). The formation of M-S-H is likely to alter the properties of the cement matrix because it is linked to the decalcification of C-S-H. However, relatively few data on M-S-H exist in the literature. In order to characterize, physically and mechanically, the M-S-H phase, pure M-S-H cohesive pastes are needed. This work studies the formation of cohesive M-S-H pastes made with MgO-to-SiO Magnesium (Mg) enrichment of cementitious materials has been observed in civil engineering construction exposed to three types of aqueous environments: seawater, soft water 2 or CH in cement notation) and calcium silicate hydrates (C-S-H) in particular—to form brucite and magnesium silicate hydrates (M-S-H). For ordinary binders with a high calcium-to-silicon ratio (Ca/Si), i.e., Portland cements, Mg attacks have been evidenced by the formation of brucite from the dissolution of portlandite. Brucite precipitates first at the surface of concrete specimens, reducing the kinetics of further species diffusion. In the long term, a slow decalcification of C-S-H leads to the formation of M-S-H  CuKα. 2 was quantified from the weight loss around 420 °C (335–455 °C) using the stepwise method  CuKα) were present in the sample cured at 50 °C. The same observations were made for the other M/S pastes tested in the present study.The sample cured at 20 °C contained brucite and amorphous silica a. The prPastes prepared with colloidal silica. Mg and Si EDS mapping of M-S-H pastes with M/S = 0.78 and colloidal silica are shown in In conclusion, the thermal curing at 50 °C resulted in a more homogeneous paste. Pastes showed a lower content of brucite at this temperature than pastes cured at 20 °C. No brucite was observed for the pastes with an M/S ratio equal to 0.78, even after only 16 days of curing for pastes designed with colloidal silica b.To produce M-S-H with different M/S ratios, with properties comparable to those observed in synthesis (on powders) ,33,43 anThe kinetics of M-S-H formation at 20 °C, 50 °C and 70 °C have been studied on M-S-H powders by Bernard . The purThe precipitation of brucite was not impeded by curing at 50 °C for the two sources of silica. Brucite was observed on the XRD for M/S = 1.3 after 19 days of hydration at 50 °C  shift thermodynamic equilibria. Bernard [2 dissolving and diffusing over time. The ratio in solution decreased, becoming more favorable to M-S-H and less to brucite (which dissolved).The effect of thermal curing could be twofold in that it may: (i) accelerate the dissolution of SiO Bernard  stated t2  is a slow process that can be accelerated by thermal curing. As expected, the higher the M/S ratio, the longer the brucite is present.In conclusion, the formation of M-S-H pastes from MgO and SiOAs detailed in the previous section, the protocol with colloidal silica and a 50 °C curing yielded the highest content of M-S-H and the lowest content of other species. In this section, the samples analyzed originate from this protocol exclusively. Two types of mixing were tested, mechanical and manual. Three M/S ratios were studied: 0.78, 1 and 1.3. The pastes considered for The morphology of M-S-H pastes was observed by SEM on fractured samples , provingX-ray diffractograms of three M-S-H pastes are presented in 2O (poorly bounded) loss in M-S-H.The TGA curves of M-S-H pastes and the derivative weight losses are shown in 2+, while the one between 750 °C and 840 °C would be linked to silanol groups on M-S-H. In the case of synthetic talc, Dumas et al. [The second and third water losses have been attributed to silanol and/or magnesium hydroxyl groups in M-S-H, but they may also comprise water present as a monolayer on the M-S-H surface ,43,52. Ts et al.  attributs et al.  on amorpThe position shared by the authors quoted is the following: (i) the weight losses between 200 °C and 800 °C for M-S-H systems are linked to the dehydroxylation of a hydroxyl group in M-S-H, and (ii) the potential offsets of the losses are linked to the evolution of the structure of M-S-H. With regard to the type of hydroxyl groups associated with each temperature, the most recent and documented assumption  was choThe second water loss region between 280 °C and 750 °C differed with the M/S ratio . For M/S2+ ions [2O and hydroxyl groups [The structure of M-S-H has been compared to that of many phyllosilicates  and characterized as a sheet structure evolving with the M/S ,35,38,43l groups . AccordiThe two analyses allowed to describe the pastes as composed only of M-S-H. The evolution of the structure with M/S appeared in the XRD and TGA. Talc-like characteristics were observed at low M/S, while serpentine-like reflections were associated with high M/S. Moreover, we detected an increase in the Mg–OH content of M-S-H as M/S increased.Several techniques were used to characterize the microstructure and porosity of M-S-H pastes. 2 adsorption-desorption isotherms of M-S-H samples made with M/S = 0.78, M/S = 1.0 and M/S = 1.3, mixed manually . The internal structure of M-S-H remained the same since low pressures were not affected.manually b or mechmanually a. The mi0 was characteristic of the presence of microporosity .0) which would correspond to H2 type. The Type H4 loop is associated with narrow slit-like pores while the Type H2 is harder to interpret and is often associated with pore size and a not well-defined shape [2O/SiO2 = 50 by Bisio et al. [Hysteresis has been associated with capillary condensation taking place in mesopores (2–50 nm in diameter) . M-S-H ped shape . The fored shape . Overallo et al. . The foro et al.  under hy2 adsorbed: the lower the M/S, the higher the total N2 adsorbed.According to BET) on M-S-H pastes hydrated from 2  to 7.5 months  are displayed in The specific surface areas calculated with BET , a gap arose between the data of Bernard, the data for manual mixing (w/b = 1.45) and the data for mechanical mixing (w/b = 1.1). The measurement uncertainty and the standard deviation (obtained by repeating the test) are too low to explain the difference in value between the protocols for these M/S values. High M/S ratios seem to be more affected by curing time and type of mixing. As for the effect of the mixing protocol at high M/S—highlighted in 2 implies a slower consumption of brucite. This could explain a different low-scale organization of the material between the pastes, following the initial brucite content . The consumption of brucite to form M-S-H in a hardening shape could produce a different porous network.Contrary to Bernard’s hypothesis, according to which M-S-H forms in non-diluted systems or might exhibit a smaller SSA in field experiments, the experiments presented in this paper proved that M-S-H pastes showed similar specific surfaces to powders, both cured at 50 °C. The specific surface seemed independent of the mixing protocol for low M/S ratios (0.78 and 1). The SSAext are plotted in BET, the values measured were higher than those of Bernard on powders equilibrated for 3.3 years at 20 °C [ext and pore volumes were notably high for all our samples, implying a high content of micro- and mesopores. A clear trend was visible with the evolution of the M/S ratio. An increase in M/S implied a decrease in external surface area and pore volume. The porosity of samples with high M/S ratios (M/S = 1.3) seemed to contain less micro- and mesopores than the porosity of samples with low M/S ratios (M/S = 0.78).Pore volumes and SSAat 20 °C . No valu3/g) increased as the M/S atomic ratio decreased, confirming that the M-S-H pastes with high M/S contained less micro- and mesopores than the pastes with lower M/S. The common location of the peak and the similar appearance of the curves indicated that the mixing protocol and the w/b did not influence this range of pores.The pore size distribution between 2.5 and 8 nm—according to the BJH method—is shown in 5 nm is shown in In order to characterize the pore size distribution at the higher scale, MIP was used. The pore size distribution between 8 and 4 × 10Autoradiography was useful for establishing a link between the pore distribution and the overall porosity of the material, since it provides a 2D image of void percentage on samples and a histogram of the void percentage distribution. Autoradiography analyses were carried on three samples (one for M/S = 0.78 and two for M/S = 1.3). 2 adsorbed. Thus, the difference in the cracking pattern was not due to the finer distribution of the pores. While the pastes with the lowest M/S (M/S = 0.78) were the most porous, they underwent the least cracking. Preferential and faster cracking appearing for high M/S could therefore be linked to lower mechanical properties of the material.The M/S-dependent effect on cracking was observed in the autoradiography maps. As explained in Autoradiography and MIP provided a first idea of the total open porosity of the material. Other measurements were carried out in order to obtain a clearer picture of the porosity of the pastes . The lowMIP explored only the connected porosity; while pores > 8 nm were taken into account, large defects (air bubbles and very open cracks) were ignored. The porosity values obtained by helium pycnometry correspond to the open porosity, including cracks and bubbles. Autoradiography measured the connected porosities, with air bubbles and cracks being removed during post-processing . Water porosity took into account the connected porosity, bubbles and cracks. The latter is therefore the largest measured value. Moreover, the 105 °C drying protocol used for water porosity may consume certain hydrates, resulting in a potential small overestimation of the actual porosity. As each method had its limitations, it was interesting to combine them all to characterize the material.2 physisorption and MIP. The more the M/S increases, the more the absorbed amounts of N2 and Hg and the specific surface area decrease, thus indicating a higher mesoporosity. On a larger scale, the porosities measured by the different techniques show this same trend. The pastes were mainly mesoporous, with a small amount of micropores and some defects appearing on a larger scale (bubbles due to mixing and cracks due to pre-treatments).A general trend could still be observed. Manually mixed pastes had a slightly higher porosity (10% more) than mechanically mixed ones . Overall, the porosity of these pastes was high. The influence of the M/S ratio on the overall porosity was difficult to assess because the pastes were very porous (due to the high w/b ratio imposed). C-S-H pastes cast with a similar protocol  also have a high porosity, close to 60% according to Kangni-Foli . The eff2 adsorbed, as well as a reduction of the specific surface (SSABET), the SSAext and the micro- and mesopore volume. At the level of capillary pores to clusters of globules of M-S-H  accelerated the consumption of brucite and favored the formation of M-S-H, with colloidal silica enabling a better distribution of particles than silica fume. The higher the M/S ratio, the longer the brucite was present. The protocol of choice for M-S-H manufacturing therefore involves colloidal silica and MgO with w/b = 1 and mechanical mixing (140–285 rd/min rotation for 10 min), followed by a thermal cure at 50 °C for 50 days (minimum recommended).X-ray diffraction and thermogravimetric analyses indicated a rearrangement of the structure—from talc-like structure to serpentine—as M/S increased, linked to a decrease in the interlayer distance and an increase in the Mg–OH content in M-S-H. The evolution of the M-S-H structure would be similar to that of the C-S-H. A parallel could be drawn between the Ca–OH that forms between the chains of Ca–Si and the Mg–OH that would form between the layers of Mg–Si as C/S or M/S increase.The porosity of M-S-H pastes was studied from the level of entrained air bubbles and voids to the level of silica sheets. On a large scale, the combination of different characterization methods made it possible to quantify the total, open porosities of the pastes. Mechanical mixing reduced the open porosity from 70%  to 62%. This value changed along with the M/S ratio in that the higher the ratio, the lower the porosity. On a smaller scale—from the capillary pores to the level of clusters of M-S-H globules—manual mixing resulted in an additional family of pores (around 30 nm) that mechanically mixed pastes did not have, the latter only showing a family of mesopores centered on 10 nm. The finer porosity of the mechanically mixed pastes can be explained by the reduction in w/b due to increased mixing speed and power, which leads to denser and less porous pastes. On the smallest scale studied in this article (silica sheets), it seemed that the specific surface area and the amount of porosity increased when the M/S ratio decreased, while the size of the pores remained unaltered. M-S-H comprise a highly porous phase with plate-like micro- and mesopores and are sensitive to changes in humidity.


However, there are no reports on wheat (Triticum aestivum) m6A transcriptome-wide map and its potential biological function in wheat resistance to wheat yellow mosaic virus (WYMV). To the best of our knowledge, this study is the first to determine the transcriptome-wide m6A profile of two wheat varieties with different resistances to WYMV. By analyzing m6A-sequencing (m6A-seq) data, we identified 25,752 common m6A peaks and 30,582 common m6A genes in two groups [WYMV-infected resistant wheat variety (WRV) and WYMV-infected sensitive wheat variety (WSV)], and all these peaks were mainly enriched in 3′ untranslated regions and stop codons of coding sequences. Gene Ontology analysis of m6A-seq and RNA-sequencing data revealed that genes that showed significant changes in both m6A and mRNA levels were associated with plant defense responses. Kyoto Encyclopedia of Genes and Genomes analysis revealed that these selected genes were enriched in the plant–pathogen interaction pathway. We further verified these changes in m6A and mRNA levels through gene-specific m6A real-time quantitative PCR (RT-qPCR) and normal RT-qPCR. This study highlights the role of m6A methylation in wheat resistance to WYMV, providing a solid basis for the potential functional role of m6A RNA methylation in wheat resistance to infection by RNA viruses.N Furthermore, different m6A RNA modifications in the two varieties demonstrated regulation of gene expression and pathogen-plant interaction-related pathways. Our study provides a basis for a comprehensive understanding of the roles of m6A modification in the molecular mechanisms underlying the interaction between wheat host and RNA viruses, especially WYMV.Wheat RNA viruses, especially wheat yellow mosaic virus (WYMV), cause a severe reduction in production in the major wheat-growing areas in China . WYMV isviridae) . The WYMviridae) . Wheat iviridae) . In RNA r genome . Neverth6A IP sequence. These samples Stored at −80°C for RNA extraction and IP-qPCR and m6A IP sequence.To obtain sufficient total RNA for IP of m6A-containing mRNA, ∼30 reviving stage plants each of yannong 999 and yannong 24 wheat infected or un-infected with WYMV were collected from a diseased nursery in Yantai city, Shandong province, China, respectively. WYMV-infected yannong 24 plants with typical yellow mosaic symptoms on the leaves, show stunted spring growth and reduced tillering. WYMV-infected yannong 999 plants show normal phenotypes without yellow mosaic symptoms . Each whWheat leaves were collected, frozen, and stored at −80°C until use. Total RNA was extracted from plants using Trizol reagent (Invitrogen). NanoDrop ND-1000  was used to quantify the amount and purity of RNA in each sample. RNA integrity was investigated using a Bioanalyzer 2100  with RIN number >7.0, and further confirmed through electrophoresis with denaturing agarose gel. Then, Dynabeads Oligo (dT) 25–61005  was used to purify the poly (A) RNA from 150 μg total RNA. Finally, the poly(A) RNA was randomly fragmented into small pieces using a Magnesium RNA Fragmentation Module .6A-specific antibody  in IP buffer  for 2 h at 4°C. Then, the IP RNA was reverse-transcribed to cDNA, and U-labeled second-stranded DNAs were synthesized using cDNA with E. coli DNA polymerase I, RNase H , and dUTP Solution . Then, the strands were prepared with A-base and ligated to the indexed adapters containing a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. AMPureXP beads were used to screen fragments of the right size. The U-labeled second-stranded DNAs treated with heat-labile UDG enzyme  were next amplified by PCR to generate a cDNA library with an average insert size of 300 ± 50 bp. Finally, 2 × 150 bp paired-end sequencing (PE150) was performed on an Illumina NovaseqTM 6000 , following the manufacturer’s recommended protocol.The total RNA was fragmented and then incubated with m1 to remove reads with adaptor contamination, low quality bases, and bases with undefined default parameters. HISAT2, an online software2 to map reads to the reference genome of Triticum aestivum (Version: IWGSC v1.0). Mapped reads of IP and input data were used for analysis using the R package exomePeak document3 then m6A intensity was visualized using Integrative Genomics Viewer (IGV) software4. MEME and HOMER56 were used for motif analysis. Called peaks were annotated by intersection with gene architecture using the R package ChIP seeker. The expression levels of all mRNAs from the input libraries were determined using StringTie7 by calculating FPKM [total exon fragment/mapped reads (millions) × exon length (kB)]. Differentially expressed genes were screened out using the criteria: fold change >1.5 or fold change <0.5, and p-value < 0.05 by the R package edgeR8.The raw data were processed by the online FASTP software6A antibody-coupled beads, while a portion of fragmented RNA was used as an input control. After ethanol precipitation, the input RNA and immunoprecipitated RNA were eluted from the beads. Both input control and m6A-IP samples were subjected to real-time quantitative PCR (RT-qPCR) with gene-specific primers. RT-PCR analysis was performed using an ABI Q5 Sequence Detection System  with AceQ qPCR SYBR Green Master Mix . At least, three biological replicates, with three technical replicates, were used for each assay. The T. aestivum cell division cycle (TaCDC) gene (Accession Number: XM_020313450) was used as the internal reference gene for analysis to calculate the fold changes in gene expression. The fold changes were calculated using the 2–ΔΔC(t) method (6A levels was performed as previously described (C(t)–ΔΔ. Statistical analyses were performed done using the Student’s t-test. Asterisks indicate a significant difference when compared to the control. ∗p < 0.05; ∗∗p < 0.01.Total RNA was extracted and fragmented into 300-nucleotide-long fragments and incubated with anti-m) method . The calescribed . In brieCP-specific primers. The results assay showed that the accumulation of WYMV was significantly increased in WSV than that in WRV (6A-seq and RNA-sequencing (RNA-seq) using the Illumina NovaseqTM 6000. A total of 129–139 million reads were acquired from the m6A-seq dataset, and 124–136 million reads were obtained from the RNA-seq dataset. After statistical analysis and quality control, there were still 100–135 million and 123–133 million valid reads in the m6A IP-seq and RNA-seq datasets, respectively. Indeed, the reads with a sequencing error rate <0.1% were more than 92% (Q30 > 92%), indicating that our data were clean (T. aestivum) (Version: IWGSC v1.0) reference genome, more than 90% of the reads in the m6A IP-seq dataset were mapped. Moreover, almost 70% of the valid reads were uniquely mapped reads, similar to the results of a previous study on rice  of the two groups were identified by comparing the mapped reads of IP and input  using the R package, exomePeak. There were 45,067 m6A peaks in the WRV group and 37,718 m6A peaks in the WSV group. Moreover, the m6A peaks in the WRV and WSV groups represented transcripts of 35,993 and 35,649 genes, respectively  had only one or two m6A-modified sites. However, genes with two m6A-modified sites in the WRV group (7.8%) were more than those in the WSV group (5.3%), and the WRV group unique m6A genes had more methylated sites than those of the WSV group   . Further < 0.05) .6A-modified genes are involved, Gene Ontology (GO) enrichment analysis of these differentially methylated genes (DMGs) was performed. The results showed that the DMGs were mainly enriched in DNA transcription, protein post-translational modification (PTM), stress response including DNA-templated transcription, protein phosphorylation, ubiquitin-dependent protein catabolic process and defense response, respectively  pathway analysis was performed, revealing that the majority of the DMGs were enriched in plant hormone signal transduction and plant–pathogen interaction pathways   . Subsequ < 0.05) . To furt < 0.05) . Therefo6A RNA modifications are related to gene expression , and the results were displayed using a four-quadrant diagram, with the following four parts: hypo-up and hypo-down, and hyper-up and hyper-down, representing hypo/hyper m6A modifications causing gene expression up/down regulation, respectively. There were 729 differentially genes, among which 347 genes belonged to the hypo-up, 104 genes to the hyper-up, 228 genes to the hypo-down, and 50 genes to the hyper-down quadrants  among the central proteins shown in the protein interaction network with complicated interaction relationship, and two gene (TraesCS1B02G175900 and TraesCS2D02G321400) related to both plant–pathogen interaction and plant hormone signal transduction pathways were randomly selected for gene-specific m6A qPCR and normal RT-qPCR. To eliminate the effect from the different genotypes of two wheat varieties to our result, we first detected the m6A levels and mRNA expression of these selected genes in two healthy wheat variety plants. Then the results displayed that most genes had no significant changes either in m6A levels or mRNA levels between two varieties -activated signaling pathway  encoding a writer protein TaFIP37-1 and another gene (TraesCS4D02G261812) encoding an eraser protein TaALKBH29B both exhibited abnormal expression according to our RNA-seq data , a homologous gene of TraesCS7B02G4 46900.1, is downregulated under dithiothreitol -induced endoplasmic reticulum stress, and thus OsGRP94 may participate in ER stress-induced autophagy and programmed cell death  BioProject, JY and JC conceived the project and designed the experiments. TZ and JY carried out the experiments with assistance from ZW, ZC, HH, PL, SG, FZ, LH, MX, and PJ. JY, TZ, and JC wrote the manuscript. All authors analyzed and discussed the results.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.