The Resistance of Soybean Variety Heinong 84 to Apple Latent Spherical Virus Is Controlled by Two Genetic Loci.

Apple latent spherical virus (ALSV) is widely used as a virus-induced gene silencing (VIGS) vector for function genome study. However, the application of ALSV to soybeans is limited by the resistance of many varieties. In this study, the genetic locus linked to the resistance of a resistant soybean variety Heinong 84 was mapped by high-throughput sequencing-based bulk segregation analysis (HTS-BSA) using a hybrid population crossed from Heinong 84 and a susceptible variety, Zhonghuang 13. The results showed that the resistance of Heinong 84 to ALSV is controlled by two genetic loci located on chromosomes 2 and 11, respectively. Cleaved amplified polymorphic sequence (CAPS) markers were developed for identification and genotyping. Inheritance and biochemical analyses suggest that the resistance locus on chromosome 2 plays a dominant dose-dependent role, while the other locus contributes a secondary role in resisting ALSV. The resistance locus on chromosome 2 might encode a protein that can directly inhibit viral proliferation, while the secondary resistance locus on chromosome 11 may encode a host factor required for viral proliferation. Together, these data reveal novel insights on the resistance mechanism of Heinong 84 to ALSV, which will benefit the application of ALSV as a VIGS vector.

Acidovorax citrulli type III effector AopU interferes with plant immune responses and interacts with a watermelon E3 ubiquitin ligase.

Acidovorax citrulli is a seed-borne bacterium that causes bacterial fruit blotch of watermelon and other cucurbit plants worldwide. It uses a type III secretion system to inject type III effectors (T3Es) into plant cells, which affect the host immune responses and facilitate pathogen colonization. However, the current understanding of the specific molecular mechanisms and targets of these effectors in A. citrulli is limited. In this study, we characterized a novel T3E called AopU in A. citrulli group II strain Aac5, which shares homology with XopU in Xanthomonas oryzae. The Agrobacterium-mediated gene transient expression system was used to study the effect of AopU on host immunity. The results showed that AopU localized on the cell membrane and nucleus of Nicotiana benthamiana, inhibited reactive oxygen species burst induced by flg22 and the expression of marker genes associated with pathogen-associated molecular pattern-triggered immunity, but activated salicylic acid and jasmonic acid signal pathways. Further investigations revealed that AopU interacts with E3 ubiquitin ligase ClE3R in watermelon, both in vitro and in vivo. Interestingly, the deletion of aopU did not affect the virulence of A. citrulli, suggesting that AopU may have functional redundancy with other effectors in terms of its role in virulence. Collectively, these findings provide new insights into the mechanism of plant immune responses regulated by A. citrulli T3Es.

pilA Gene Contributes to Virulence, Motility, Biofilm Formation, and Interspecific Competition of Bacteria in Acidovorax citrulli.

Acidovorax citrulli, the causative agent of bacterial fruit blotch, can be divided into two main groups based on factors such as pathogenicity and host species preference. PilA is an important structural and functional component of type IV pili (T4P). Previous studies have found significant differences in pilA DNA sequences between group I and group II strains of A. citrulli. In this study, we characterized pilA in the group I strain pslb65 and the group II strain Aac5. pilA mutants, complementation strains, and cross-complementation strains were generated, and their biological phenotypes were analyzed to identify functional differences between pilA in the two groups. pilA deletion mutants (pslb65-ΔpilA and Aac5-ΔpilA) showed significantly reduced pathogenicity compared with the wild-type (WT) strains; pslb65-ΔpilA also completely lost twitching motility, whereas Aac5-ΔpilA only partially lost motility. In King's B medium, there were no significant differences in biofilm formation between pslb65-ΔpilA and WT pslb65, but Aac5-ΔpilA showed significantly reduced biofilm formation compared to WT Aac5. In M9 minimal medium, both mutants showed significantly lower biofilm formation compared to the corresponding WT strains, although biofilm formation was recovered in the complementation strains. The biofilm formation capacity was somewhat recovered in the cross-complementation strains but remained significantly lower than in the WT strains. The interspecies competitive abilities of pslb65-ΔpilA and Aac5-ΔpilA were significantly lower than in the WT strains; Aac5-ΔpilA was more strongly competitive than pslb65-ΔpilA, and the complementation strains recovered competitiveness to WT levels. Furthermore, the cross-complementation strains showed stronger competitive abilities than the corresponding WT strains. The relative expression levels of genes related to T4P and the type VI secretion system were then assessed in the pilA mutants via quantitative PCR. The results showed significant differences in the relative expression levels of multiple genes in pslb65-ΔpilA and Aac5-ΔpilA compared to the corresponding WT stains. This indicated the presence of specific differences in pilA function between the two A. citrulli groups, but the regulatory mechanisms involved require further study.

Orientin inhibits the progression of fibroblast-like synovial cells in rheumatoid arthritis by regulating MAPK-signaling pathway.

BACKGROUND: Natural compounds are found to play an essential role in diverse inflammatory diseases, including rheumatoid arthritis (RA). Orientin, a flavonoid compound, is closely related to diverse pathological processes. Nevertheless, the role of orientin in RA is still unknown. METHODS: The cell viability was tested through cell counting kit 8 (CCK-8) assay, and the number of cell colonies was calculated via colony formation assay. In addition, flow cytometry assay was employed to detect apoptosis rate in human RA fibroblast-like synoviocytes (RA-FLS). Besides, Transwell assay was introduced to determine cell migratory and invasive abilities. Moreover, the level of cytokines (IL-8, IL-1ß, and IL-6) was estimated with quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent serologic assay. Furthermore, western blotting analysis was used to test the protein levels of cleaved-caspase-3, Bax, BCL-2, matrix metalloproteinase (MMP)-2, MMP-9, phosphorylated c-Jun N-terminal kinase, p-P38, and phospho-extracellular signal-related kinase. RESULTS: Orientin inhibited cell viability, migration as well as invasion in a concentration- dependent manner in human RA-FLS. Additionally, treatment of orientin facilitated apoptosis and decreased the secretion of cytokines induced by tumor necrosis factor alpha (TNF-α) in human RA-FLS. Moreover, orientin inactivated mitogen-activated protein kinase (MAPK)-related signaling pathway, notably in human RA-FLS. CONCLUSION: These findings confirmed that orientin inhibited human RA-FLS development and decreased TNF-α-induced inflammatory factors, at least partly, by modulating MAPK-signaling pathway, which implied that orientin might be an effective agent for treating RA.

Acidovorax citrulli Effector AopV Suppresses Plant Immunity and Interacts with Aromatic Dehydratase ADT6 in Watermelon.

Bacterial fruit blotch (BFB) is a disease of cucurbit plants caused by Acidovorax citrulli. Although A. citrulli has great destructive potential, the molecular mechanisms of pathogenicity of A. citrulli are not clear, particularly with regard to its type III secreted effectors. In this study, we characterized the type III secreted effector protein, AopV, from A. citrulli strain Aac5. We show that AopV significantly inhibits reactive oxygen species and the expression of PTI marker genes, and helps the growth of Pseudomonas syringae D36E in Nicotiana benthamiana. In addition, we found that the aromatic dehydratase ADT6 from watermelon was a target of AopV. AopV interacts with ADT6 in vivo and in vitro. Subcellular localization indicated ADT6 and AopV were co-located at the cell membrane. Together, our results reveal that AopV suppresses plant immunity and targets ADT6 in the cell membrane. These findings provide an new characterization of the molecular interaction of A. citrulli effector protein AopV with host cells.

Hcp of the Type VI Secretion System (T6SS) in Acidovorax citrulli Group II Strain Aac5 Has a Dual Role as a Core Structural Protein and an Effector Protein in Colonization, Growth Ability, Competition, Biofilm Formation, and Ferric Iron Absorption.

A type VI secretion system (T6SS) gene cluster has been reported in Acidovorax citrulli. Research on the activation conditions, functions, and the interactions between key elements in A. citrulli T6SS is lacking. Hcp (Hemolysin co-regulated protein) is both a structural protein and a secretion protein of T6SS, which makes it a special element. The aims of this study were to determine the role of Hcp and its activated conditions to reveal the functions of T6SS. In virulence and colonization assays of hcp deletion mutant strain Δhcp, tssm (type VI secretion system membrane subunit) deletion mutant strain Δtssm and double mutant ΔhcpΔtssm, population growth was affected but not virulence after injection of cotyledons and seed-to-seedling transmission on watermelon. The population growth of Δhcp and Δtssm were lower than A. citrulli wild type strain Aac5 of A. citrulli group II at early stage but higher at a later stage. Deletion of hcp also affected growth ability in different culture media, and the decline stage of Δhcp was delayed in KB medium. Biofilm formation ability of Δhcp, Δtssm and ΔhcpΔtssm was lower than Aac5 with competition by prey bacteria but higher in KB and M9-Fe3+ medium. Deletion of hcp reduced the competition and survival ability of Aac5. Based on the results of Western blotting and qRT-PCR analyses, Hcp is activated by cell density, competition, ferric irons, and the host plant. The expression levels of genes related to bacterial secretion systems, protein export, and several other pathways, were significantly changed in the Δhcp mutant compared to Aac5 when T6SS was activated at high cell density. Based on transcriptome data, we found that a few candidate effectors need further identification. The phenotypes, activated conditions and transcriptome data all supported the conclusion that although there is only one T6SS gene cluster present in the A. citrulli group II strain Aac5, it related to multiple biological processes, including colonization, growth ability, competition and biofilm formation.

Essential Acidovorax citrulli Virulence Gene hrpE Activates Host Immune Response against Pathogen.

Bacterial fruit blotch (BFB) caused by Acidovorax citrulli (Ac) is a devastating watermelon disease that severely impacts the global watermelon industry. Like other Gram-negative bacteria, the type three secretion system (T3SS) is the main pathogenicity factor of A. citrulli. The T3SS apparatus gene hrpE codes for the Hrp pilus and serves as a conduit to secret effector proteins into host cells. In this study, we found that the deletion of hrpE in A. citrulli results in the loss of pathogenicity on hosts and the hypersensitive response on non-hosts. In addition, the A. citrulli hrpE mutant showed a reduction in in vitro growth, in planta colonization, swimming and twitching motility, and displayed increases in biofilm formation ability compared to the wild type. However, when HrpE was transiently expressed in hosts, the defense responses, including reactive oxygen species bursts, callose deposition, and expression of defense-related genes, were activated. Thus, the A. Citrulli growth in HrpE-pretreated hosts was suppressed. These results indicated that HrpE is essential for A. citrulli virulence but can also be used by hosts to help resist A. citrulli. Our findings provide a better understanding of the T3SS pathogenesis in A. citrulli, thus providing a molecular basis for biopesticide development, and facilitating the effective control of BFB.

Methyl-CpG-binding protein 2 promotes osteogenic differentiation of bone marrow mesenchymal stem cells through regulating forkhead box F1/Wnt/ß-Catenin axis.

Postmenopausal osteoporosis is characterized by inadequate bone formation of osteoblasts and excessive bone resorption of osteoclasts. Bone marrow mesenchymal stem cells (BMSCs), with the potential of osteogenic differentiation, have been widely used in the bone tissues engineering for the treatment of bone diseases, including postmenopausal osteoporosis. Methyl-CpG-binding protein 2 (MECP2) has been reported to be implicated in bone formation during the development of Rett syndrome. However, the influence of MeCP2 on osteogenic differentiation of BMSCs during osteoporosis remains unclear. Firstly, mice model with estrogen deficiency-induced osteoporosis was established through ovariectomy (OVX). MeCP2 was found to be down-regulated in bone tissues and BMSCs of OVX-induced osteoporosis mice. Secondly, over-expression of MeCP2 enhanced the calcium deposition of BMSCs isolated from the OVX-induced osteoporosis mice. Moreover, expression of osteogenic biomarkers including alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), collagen type I alpha 1 (COL1A1), and osteocalcin (OCN) was increased in BMSCs by overexpression of MeCP2. Thirdly, over-expression of MeCP2 reduced protein expression of forkhead box F1 (FOXF1) and adenomatous polyposis coli (APC), while enhanced Wnt5a and ß-catenin expression in BMSCs. Over-expression of FOXF1 attenuated MeCP2 over-expression-induced decrease of FOXF1 and APC, as well as increase of Wnt5a and ß-catenin. Finally, the increased calcium deposition, protein expression of ALP, RUNX2COL1A1 and OCN induced by concomitant overexpression of MeCP2 were also restored by FOXF1 over-expression. In conclusion, MeCP2 promoted osteogenic differentiation of BMSCs through regulating FOXF1/Wnt/ß-Catenin axis to attenuate osteoporosis. MeCP2 over-expression reduced FOXF1 to promote the activation of Wnt5a/ß-Catenin and promote osteogenic differentiation of BMSCs during the prevention of postmenopausal osteoporosis.

Radiofrequency Ablation of Parathyroid Glands to Treat a Patient With Hypercalcemia Caused by a Novel Inactivating Mutation in CaSR.

Objective: We identified a novel inactivating mutation in the calcium-sensing receptor (CaSR) gene in a patient with refractory hypocalciuric hypercalcemia and analyzed its function. The effectiveness of radiofrequency ablation of the parathyroid glands to treat hypercalcemia caused by this mutation was explored. Methods: Clinical data of patients before and after radiofrequency ablation were retrospectively analyzed. The CaSR mutation (D99N) found in the patient was studied in cell lines. HEK-293 cells were transfected with plasmids containing wild-type (WT) or mutant CaSR genes (D99N and W718X). Expression levels of the respective CaSR proteins were measured, and their functions were assessed by examining the effect of NPS R-568 (a CaSR agonist) on intracellular Ca2+ oscillations and that of exogenous parathyroid hormone (PTH) on intracellular cyclic adenosine monophosphate (cAMP) levels. Results: The effectiveness of pharmacological treatment was poor, whereas radiofrequency ablation of the parathyroid glands resulted in controlled blood calcium and PTH levels in the patient. In cell lines, upon NPS R-568 administration, the amplitude of intracellular Ca2+ oscillations in the D99N group was lower than that in the WT group and higher than that in the W718X group. Upon administration of PTH, intracellular cAMP levels in the D99N group were higher than those in the WT group and lower than those in the W718X group. Conclusion: The homozygous mutation D99N reduced CaSR activity and caused more severe hypocalciuric hypercalcemia. For patients with this type of hypercalcemia and poor response to pharmacological treatments, radiofrequency ablation of the parathyroid glands may be a suitable treatment option.

Combined 17α-hydroxylase/17,20-lyase deficiency with short stature: case study.

BACKGROUNDS: 17α-hydroxylase/17,20-lyase deficiency (17-OHD) is an uncommon form of congenital adrenal hyperplasia. Most patients are tall owing to delayed closure of epiphyses as a result of deficiency of sex hormones. METHODS: We present a 17-OHD case with unusual short stature and reviewed related literature. RESULTS: A 17-year-old female patient presented with primary amenorrhea, hypertension, hypokalemia and hypergonadotropic hypogonadism (HH). Sequencing of the CYP17A1 gene identified a homozygous c.985_987delTACinsAA in exon 6 that confirmed the diagnosis of 17-OHD. However, her height (148 cm, height standard deviation score [HSDS] -2.28) was unusually low compared with that of other 17-OHD patients. Levels of growth hormone (GH) and insulin-like growth factor (IGF)-1 were normal, and the GH provocation test excluded the possibility of GH deficiency. She underwent glucocorticoid and sex-hormone replacement therapy, reaching a final height of 152 cm (HSDS -1.59). These data suggest that tall stature is not a requisite characteristic of 17-OHD. Further studies are needed to clarify the effects of sex hormone on linear bone growth (LBG) in 17-OHD patients.