A rust fungus effector directly binds plant pre-mRNA splice site to reprogram alternative splicing and suppress host immunity.

Alternative splicing (AS) is a crucial post-transcriptional regulatory mechanism in plant resistance. However, whether and how plant pathogens target splicing in their host remains mostly unknown. For example, although infection by Puccinia striiformis f. sp. tritici (Pst), a pathogenic fungus that severely affects the yield of wheat worldwide, has been shown to significantly influence the levels of alternatively spliced transcripts in the host, the mechanisms that govern this process, and its functional consequence have not been examined. Here, we identified Pst_A23 as a new Pst arginine-rich effector that localizes to host nuclear speckles, nuclear regions enriched in splicing factors. We demonstrated that transient expression of Pst_A23 suppresses plant basal defence dependent on the Pst_A23 nuclear speckle localization and that this protein plays an important role in virulence, stable silencing of which improves wheat stripe rust resistance. Remarkably, RNA-Seq data revealed that AS patterns of 588 wheat genes are altered in Pst_A23-overexpressing lines compared to control plants. To further examine the direct relationship between Pst_A23 and AS, we confirmed direct binding between two RNA motifs predicted from these altered splicing sites and Pst_A23 in vitro. The two RNA motifs we chose occur in the cis-element of TaXa21-H and TaWRKY53, and we validated that Pst_A23 overexpression results in decreased functional transcripts of TaXa21-H and TaWRKY53 while silencing of TaXa21-H and TaWRKY53 impairs wheat resistance to Pst. Overall, this represents formal evidence that plant pathogens produce 'splicing' effectors, which regulate host pre-mRNA splicing by direct engagement of the splicing sites, thereby interfering with host immunity.

Abnormal DNA methylation in oocytes could be associated with a decrease in reproductive potential in old mice.

PURPOSE: This study was designed to evaluate DNA methylation and the expression of DNA methyltransferases (Dnmt1, Dnmt3a, Dnmt3b and Dnmt3L) in metaphaseII (MII) oocytes and the DNA methylation of pre-implantation embryos during mouse aging to address whether such aging-related changes are associated with decreased reproductive potential in aged mice. METHODS: Oocytes (MII) from 6 to 8 weeks old female mice are referred to as the 'young group'; oocytes from the same group that were maintained until 35-40 weeks old are referred to as the 'old group.' The oocytes were fertilized both in vitro and in vivo to obtain embryos. The DNA methylation levels in the oocytes (MII) and pre-implantation embryos were assessed using fluorescence staining. The expression levels of the Dnmt genes in the oocytes (MII) were assessed using Western blotting. RESULTS: The DNA methylation levels in the oocytes and pre-implantation embryos (in vivo and in vitro) decreased significantly during the aging of the mice. The expression levels of all of the examined Dnmt proteins in the old group were lower than young group. Both the cleavage and blastocyst rate were significantly lower in the oocytes of the older mice (69.9 % vs. 80.9 %, P < 0.05; 33.9 % vs. 56.4 %, P < 0.05). The pregnancy rate of the old mice was lower than that of the young mice (46.7 % vs. 100 %, P < 0.05). The stillbirth and fetal malformation rate was significantly higher in the old group than in the young group (17.2 % vs. 2.9 %, P < 0.05). CONCLUSIONS: The decreased expression of Dnmt1, Dnmt3a, Dnmt3b and Dnmt3L in oocytes (MII) and the change of genome-wide DNA methylation in oocytes and pre-implantation embryos due to aging may be related to lower reproductive potential in old female mice.

Pharmacodynamics of Dracorhodin Perchlorate and Its Inflammation-Targeting Emulsion Gel for Wound Healing.

The mechanism of dracorhodin perchlorate for the repair of rat skin wounds was investigated. In order to screen a more favorable drug delivery system for wound repair, the therapeutic effect of dracorhodin perchlorate inflammation-targeted emulsion gel was compared with that of non-targeted emulsion gel on rat wounds. Compared with non-targeted emulsion gels, inflammation-targeted emulsion gels had a better transdermal penetration and lower potentials (-51.6 mV and -17.1 mV, respectively). The recovery of the wound from the dracorhodin perchlorate inflammation targeted emulsion gel group was better than that of the dracorhodin perchlorate inflammation non-targeted emulsion gel group and the positive drug group. Compared with the no-target emulsion gel group, the bFGF expression on day 7 and the EGF expression on day 14 in the targeted emulsion group showed 45.5% and 49.9% improvement, respectively. Pathological tissue slices showed that the epidermis, dermis, and basal layer inflammatory cells in the inflammation-targeted emulsion gel group and non-targeted emulsion gel group were significantly reduced, the granulation tissue proliferation was obvious, and the inflammation-targeted emulsion gel group was more effective. The results proved that dracorhodin perchlorate had a repairing effect on rat skin wounds, and its mechanism might be related to the promotion of the expression of EGF and bFGF in tissues.

Spermatozoa cryopreservation alters pronuclear formation and zygotic DNA demethylation in mice.

This study was conducted to investigate the effects of spermatozoa cryopreservation on DNA demethylation in mouse zygotes. Global methylation was studied in zygotes fertilized with cryopreserved sperm by immunostaining, and relative transcript abundance of Tet3, a key gene responsible for zygotic DNA demethylation, was examined by real-time quantitative polymerase chain reaction. Fresh sperm group served as control. Results indicated spermatozoa cryopreservation decreased fertilization rate (68.2% vs. 86.9%; P < 0.01) and delayed pronuclear formation (P < 0.05), compared with the control group. The percentages of embryos developed to cleavage and blastocyst stages in the freezing group (52.9% and 66.8%, respectively) were lower (P < 0.01 and P < 0.05, respectively) than those of the control group (83.4% and 81.1%, respectively). Furthermore, embryos obtained from cryopreserved sperm had higher relative methylation levels (P < 0.05) and less Tet3 mRNA concentrations (P < 0.01) in advanced pronuclear stages. Hence, we reported that spermatozoa cryopreservation disturbed the Tet3-mediated DNA demethylation progression in the zygotic paternal genome, which could be detrimental to the development of early mouse embryos, and most of the differences observed might be explained by delayed fertilization when using cryopreserved sperm.

Expression and distribution of cell adhesion-related proteins in bovine parthenogenetic embryos: The effects of oocyte vitrification.

The objective was to investigate expression of cell adhesion-related proteins (E-cadherin, ß-catenin, and the cytoskeletal protein F-actin) in bovine parthenogenetic embryos derived from vitrified-warmed oocytes. Bovine oocytes at metaphase II were randomly allocated into three groups: (1) untreated (control); (2) exposed to vitrification solution without freezing (toxicity); and (3) vitrified and warmed by the open-pulled straw method (vitrification). After parthenogenetic activation, in the vitrification group compared with the control, the timing of compaction was delayed in (108-120 vs. 96-108 hours, respectively), and the percentage of blastocysts that developed from eight-cell embryos was lower (32.08% vs. 61.03%; P < 0.05). To investigate whether vitrification delayed embryo compaction by affecting adhesion junction formation and function, immunostaining and quantitative reverse transcription polymerase chain reaction were done to characterize distribution patterns (E-cadherin, ß-catenin, and the cytoskeletal protein F-actin) and expression levels of cell adhesion-related proteins (ß-catenin). Distribution of ß-catenin in eight-cell embryos from the vitrification group changed dramatically compared with the control and toxicity groups. Relative expression of ß-catenin at the mRNA and protein levels was lower (P < 0.05) than that of the fresh and toxicity groups. However, expression and distribution of E-cadherin were similar among groups. In conclusion, abnormal distribution and decreased expression of ß-catenin in bovine parthenogenetic eight-cell embryos derived from vitrified-warmed oocytes were associated with embryo compaction and reduced competence for subsequent embryo development.