Partially knocking out NtPDK1a/1b/1c/1d simultaneously in Nicotiana tabacum using CRISPR/CAS9 technology results in auxin-related developmental defects.

The eukaryotic AGC protein kinase subfamily (protein kinase A/ protein kinase G/ protein kinase C-family) is involved in regulating numerous biological processes across kingdoms, including growth and development, and apoptosis. PDK1(3-phosphoinositide-dependent protein kinase 1) is a conserved serine/threonine kinase in eukaryotes, which is both a member of AGC kinase and a major regulator of many other downstream AGC protein kinase family members. Although extensively investigated in model plant Arabidopsis, detailed reports for tobacco PDK1s have been limited. To better understand the functions of PDK1s in tobacco, CRISPR/CAS9 transgenic lines were generated in tetraploid N. tabacum, cv. Samsun (NN) with 5-7 of the 8 copies of 4 homologous PDK1 genes in tobacco genome (NtPDK1a/1b/1c/1d homologs) simultaneously knocked out. Numerous developmental defects were observed in these NtPDK1a/1b/1c/1d CRISPR/CAS9 lines, including cotyledon fusion leaf shrinkage, uneven distribution of leaf veins, convex veins, root growth retardation, and reduced fertility, all of which reminiscence of impaired polar auxin transport. The severity of these defects was correlated with the number of knocked out alleles of NtPDK1a/1b/1c/1d. Consistent with the observation in Arabidopsis, it was found that the polar auxin transport, and not auxin biosynthesis, was significantly compromised in these knockout lines compared with the wild type tobacco plants. The fact that no homozygous plant with all 8 NtPDK1a/1b/1c/1d alleles being knocked out suggested that knocking out 8 alleles of NtPDK1a/1b/1c/1d could be lethal. In conclusion, our results indicated that NtPDK1s are versatile AGC kinases that participate in regulation of tobacco growth and development via modulating polar auxin transport. Our results also indicated that CRISPR/CAS9 technology is a powerful tool in resolving gene redundancy in polyploidy plants.

Knocking out NtSARD1a/1b/1c/1d by CRISPR/CAS9 technology reduces the biosynthesis of salicylic acid (SA) and compromises immunity in tetraploid Nicotiana tabacum.

Salicylic acid (SA) is a key phyto-hormone that is essential for plant immunity. SARD1 (SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1), a member of the CBP60 (CALMODULIN-BINDING PROTEIN60) gene family, is one of the major transcription factors regulating the expression of the genes in SA biosynthesis. SARD1 has been extensively studied in model plant Arabidopsis. However, the function of SARD1 homologues in SA biosynthesis and immune responses have rarely been investigated in other plant species. In this study, the CRISPR/CAS9 (Clustered Regularly Interspersed Short Palindromic Repeats/CAS9) technology was used in creating transgenic tobacco mutant lines with 6-8 alleles of four NtSARD1 homologous genes (NtSARD1a/1b/1c/1d) knocked out. No significant difference in morphological phenotype was observed between the transgenic knockout lines and the wild type tobacco plants, indicating that knocking out NtSARD1s does not affect the growth and development in tobacco. However, knocking out or partially knocking out of NtSARD1a/b/c/d resulted in a significantly reduced expression of NtICS1, the key gene in SA biosynthesis pathway, and thus the subsequently decreased SA/SAG accumulations in response to Pst DC3000 (Pseudomonas syrangae pv.tomato DC3000) infection, indicating a key role of NtSARD1 genes in SA biosynthesis in tobacco. As a consequence of reduced SA/SAG accumulation, the Pst DC3000-induced expression of NtPR genes as well as the resistance to Pst DC3000 were both significantly reduced in these knockout lines compared with the wild type tobacco plants. Interestingly, the reductions in the SA/SAG level, NtPR gene induction and Pst DC3000 resistance were positively correlated with the number of alleles being knocked out. Furthermore, LUC reporter gene driven by the promoter of NtICS1 containing two G(A/T)AATT(T/G) motifs could be activated by NtSARD1a, suggesting that NtSARD1a could bind to the core G(A/T)AATT(T/G) motifs and thus activate the expression of LUC reporter. Taken together, our results demonstrated that the NtSARD1 proteins play essential roles in SA biosynthesis and immune responses in tobacco. Our results also demonstrated that the CRISPR/CAS9 technology can overcome gene redundancy and is a powerful tool to study gene functions in polyploid plant species.

Silencing GmATG7 Leads to Accelerated Senescence and Enhanced Disease Resistance in Soybean.

Autophagy plays a critical role in nutrient recycling/re-utilizing under nutrient deprivation conditions. However, the role of autophagy in soybeans has not been intensively investigated. In this study, the Autophay-related gene 7 (ATG7) gene in soybeans (referred to as GmATG7) was silenced using a virus-induced gene silencing approach mediated by Bean pod mottle virus (BPMV). Our results showed that ATG8 proteins were highly accumulated in the dark-treated leaves of the GmATG7-silenced plants relative to the vector control leaves (BPMV-0), which is indicative of an impaired autophagy pathway. Consistent with the impaired autophagy, the dark-treated GmATG7-silenced leaves displayed an accelerated senescence phenotype, which was not seen on the dark-treated BPMV-0 leaves. In addition, the accumulation levels of both H2O2 and salicylic acid (SA) were significantly induced in the GmATG7-silenced plants compared with the BPMV-0 plants, indicating an activated immunity. Consistently, the GmATG7-silenced plants were more resistant against both Pseudomonas syringae pv. glycinea (Psg) and Soybean mosaic virus (SMV) compared with the BPMV-0 plants. However, the activated immunity in the GmATG7-silenced plant was not dependent upon the activation of MPK3/MPK6. Collectively, our results demonstrated that the function of GmATG7 is indispensable for autophagy in soybeans, and the activated immunity in the GmATG7-silenced plant is a result of impaired autophagy.

Microbiota alteration of Chinese young male adults with high-status negative cognitive processing bias.

Introduction: Evidence suggests that negative cognitive processing bias (NCPB) is a significant risk factor for depression. The microbiota-gut-brain axis has been proven to be a contributing factor to cognitive health and disease. However, the connection between microbiota and NCPB remains unknown. This study mainly sought to explore the key microbiota involved in NCPB and the possible pathways through which NCPB affects depressive symptoms. Methods: Data in our studies were collected from 735 Chinese young adults through a cross-sectional survey. Fecal samples were collected from 35 young adults with different levels of NCPB (18 individuals were recruited as the high-status NCPB group, and another 17 individuals were matched as the low-status NCPB group) and 60 with different degrees of depressive symptoms (27 individuals were recruited into the depressive symptom group, as D group, and 33 individuals were matched into the control group, as C group) and analyzed by the 16S ribosomal RNA sequencing technique. Results: As a result, the level of NCPB correlated with the degree of depressive symptoms as well as anxiety symptoms and sleep quality (p < 0.01). The ß-diversity of microbiota in young adults was proven to be significantly different between the high-status NCPB and the low-status NCPB groups. There were several significantly increased bacteria taxa, including Dorea, Christensenellaceae, Christe -senellaceae_R_7_group, Ruminococcaceae_NK4A214_group, Eggerthellaceae, Family-XIII, Family_XIII_AD3011_group, Faecalibaculum, and Oscillibacter. They were mainly involved in pathways including short-chain fatty acid (SCFA) metabolism. Among these variable bacteria taxa, Faecalibaculum was found associated with both NCPB and depressive symptoms. Furthermore, five pathways turned out to be significantly altered in both the high-status NCPB group and the depressive symptom group, including butanoate metabolism, glyoxylate and dicarboxylate metabolism, propanoate metabolism, phenylalanine, tyrosine, and tryptophan biosynthesis, valine, leucine, and isoleucine degradation. These pathways were related to SCFA metabolism. Discussion: Fecal microbiota is altered in Chinese young male adults with high status NCPB and may be involved in the biochemical progress that influences depressive symptoms.

[Nerve-sparing robot-assisted laparoscopic radical cystectomy: Clinical application and effect].

OBJECTIVE: To investigate the clinical feasibility and effect of nerve-sparing robot-assisted laparoscopic radical cystectomy (NSRA-LSRC). METHODS: We retrospectively reviewed the clinical data on 12 cases of NSRA-LSRC performed from March 2016 to May 2018. The patients were aged 45 to 65 years old and all potent before surgery, with a mean IIEF-5 score of >17. The surgical procedure involved excision of the bladder and prostate and dissection of the pelvic lymph nodes, with preservation of the bilateral neurovascular bundles, internal accessory pudendal artery and pubic bladder complex. All the patients were advised to take PDE5I postoperatively and followed up for the sexual function with the IIEF-5 scores. RESULTS: Surgical procedures were completed successfully, all with negative surgical margins. Postoperative pathology confirmed invasive high-grade urothelial carcinoma or carcinoma in situ in all the cases, including 11 cases in stage T2N0M0 or below and 1 case in stage T3aN0M0. There were no serious intraoperative or postoperative complications, nor recurrence or metastasis during the follow-up period of 12－36 (20.7 ± 8.0) months. The IIEF-5 scores of the patients at 3, 6 and 12 months after operation were 10.9 ± 6.9, 12.3 ± 6.9 and 14.1 ± 8.0, respectively. At 12 months, satisfactory sexual intercourse was achieved with the help of potency-enhancing medicine in 5 cases (41.7%), penile erection insufficient for sexual intercourse in 3 cases (25%), and no erection in 4 cases (33.3%). CONCLUSIONS: Nerve-sparing robot-assisted laparoscopic radical cystectomy can maximally preserve the sexual function of the patients with urinary bladder carcinoma.

Effect of daily average temperature on hand-foot-mouth disease incidence among children under 5 years old in Jingzhou / 中国学校卫生

Objective@#To evaluate the effect of daily temperature on hand-foot-mouth disease (HFMD) in children under 5 years old in Jingzhou city.@*Methods@#HFMD incidence data and meteorological data in Jingzhou city were obtained during 2010 and 2017. Distributed lag non-linear model (DLNM) was utilized to investigate the impact of daily temperature on HFMD incidence among children under 5 years old adjusting for potential confounders of other meteorological factors, secular trend, weekdays and holidays.@*Results@#A total of 47 525 cases were reported during 2010 to 2017, of which the ratio of male to female was 1.52. Children under 1 year old, 1-<3 years old, 3-5 years old accounted for 9.72%, 62.10%, and 28.18% of the total cases, respectively. Children cared at home and children care in kindergarten accounted for 73.29% and 26.71% of the total cases, respectively. The relationship between the temperature and the daily cases of HFMD in children under five years old was a ‘M’ pattern. Compared with a reference temperature (the 50 th percentile of average temperature during the study period, P 50), the maximum value of effect at 8.21 ℃ and 25.81 ℃ were 1.53(95%CI=1.33-1.76) and 1.47(95%CI=1.31-1.65). Higher temperatures (such as 25.81 ℃ in this paper) showed a long lag effect on the HFMD incidence compared with lower temperatures (such as 8.21 ℃). Subgroup analyses indicated that children aged 3-5 years (children who attended daycare) were more vulnerable to the effects of temperature changes on HFMD than those under 1 year old and 1-<3-year-old (cared at home).@*Conclusion@#The temperature has a significant impact on the HFMD incidence among children under five years old in Jingzhou. Daycare centers is the key place for prevention and control of HFMD.