NLR surveillance of pathogen interference with hormone receptors induces immunity.

Phytohormone signalling pathways have an important role in defence against pathogens mediated by cell-surface pattern recognition receptors and intracellular nucleotide-binding leucine-rich repeat class immune receptors1,2 (NLR). Pathogens have evolved counter-defence strategies to manipulate phytohormone signalling pathways to dampen immunity and promote virulence3. However, little is known about the surveillance of pathogen interference of phytohormone signalling by the plant innate immune system. The pepper (Capsicum chinense) NLR Tsw, which recognizes the effector nonstructural protein NSs encoded by tomato spotted wilt orthotospovirus (TSWV), contains an unusually large leucine-rich repeat (LRR) domain. Structural modelling predicts similarity between the LRR domain of Tsw and those of the jasmonic acid receptor COI1, the auxin receptor TIR1 and the strigolactone receptor partner MAX2. This suggested that NSs could directly target hormone receptor signalling to promote infection, and that Tsw has evolved a LRR resembling those of phytohormone receptors LRR to induce immunity. Here we show that NSs associates with COI1, TIR1 and MAX2 through a common repressor-TCP21-which interacts directly with these phytohormone receptors. NSs enhances the interaction of COI1, TIR1 or MAX2 with TCP21 and blocks the degradation of corresponding transcriptional repressors to disable phytohormone-mediated host immunity to the virus. Tsw also interacts directly with TCP21 and this interaction is enhanced by viral NSs. Downregulation of TCP21 compromised Tsw-mediated defence against TSWV. Together, our findings reveal that a pathogen effector targets TCP21 to inhibit phytohormone receptor function, promoting virulence, and a plant NLR protein has evolved to recognize this interference as a counter-virulence strategy, thereby activating immunity.

Interspecies/Intergroup Complementation of Orthotospovirus Replication and Movement through Reverse Genetics Systems.

Orthotospoviruses, the plant-infecting bunyaviruses, cause serious diseases in agronomic crops and pose major threats to global food security. The family of Tospoviridae contains more than 30 members that are classified into two geographic groups, American-type and Euro/Asian-type orthotospovirus. However, the genetic interaction between different species and the possibility, during mixed infections, for transcomplementation of gene functions by orthotospoviruses from different geographic groups remains underexplored. In this study, minireplicon-based reverse genetics (RG) systems have been established for Impatiens necrotic spot virus (INSV) (an American-type orthotospovirus) and for Calla lily chlorotic spot virus and Tomato zonate spot virus (CCSV and TZSV) (two representative Euro/Asian orthotospoviruses). Together with the earlier established RG system for Tomato spotted wilt virus (TSWV), a type species of the Orthotospovirus American-clade, viral replicase/movement proteins were exchanged and analyzed on interspecies transcomplementation. Whereas the homologous RNA-dependent RNA polymerase (RdRp) and nucleocapsid (N) protein supported the replication of orthotospoviruses from both geographic groups, heterologous combinations of RdRp from one group and N from the other group were unable to support the replication of viruses from both groups. Furthermore, the NSm movement protein (MP), from both geographic groups of orthotospoviruses, was able to transcomplement heterologous orthotospoviruses or a positive-strand Cucumber mosaic virus (CMV) in their movement, albeit with varying efficiency. MP from Rice stripe tenuivirus (RSV), a plant-infecting bunyavirus that is distinct from orthotospoviruses, or MP from CMV also moves orthotospoviruses. Our findings gain insights into the genetic interaction/reassortant potentials for the segmented plant orthotospoviruses. IMPORTANCE Orthotospoviruses are agriculturally important negative-strand RNA viruses and cause severe yield-losses on many crops worldwide. Whereas the emergence of new animal-infecting bunyaviruses is frequently associated with genetic reassortants, this issue remains underexposed with the plant-infecting orthotospovirus. With the development of reverse genetics systems for orthotospoviruses from different geographic regions, the interspecies/intergroup replication/movement complementation between American- and Euro/Asian-type orthotospoviruses were investigated. Genomic RNAs from American orthotospoviruses can be replicated by the RdRp and N from those of Euro/Asia-group orthotospoviruses, and vice versa. However, their genomic RNAs cannot be replicated by a heterologous combination of RdRp from one geographic group and N from another geographic group. Cell-to-cell movement of viral entity is supported by NSm from both geographic groups, with highest efficiency by NSm from viruses belonging to the same group. Our findings provide important insights into the genetic interaction and exchange ability of viral gene functions between different species of orthotospovirus.

Development of a Mini-Replicon-Based Reverse-Genetics System for Rice Stripe Tenuivirus.

Negative-stranded RNA (NSR) viruses include both animal- and plant-infecting viruses that often cause serious diseases in humans and livestock and in agronomic crops. Rice stripe tenuivirus (RSV), a plant NSR virus with four negative-stranded/ambisense RNA segments, is one of the most destructive rice pathogens in many Asian countries. Due to the lack of a reliable reverse-genetics technology, molecular studies of RSV gene functions and its interaction with host plants are severely hampered. To overcome this obstacle, we developed a mini-replicon-based reverse-genetics system for RSV gene functional analysis in Nicotiana benthamiana. We first developed a mini-replicon system expressing an RSV genomic RNA3 enhanced green fluorescent protein (eGFP) reporter [MR3(-)eGFP], a nucleocapsid (NP), and a codon usage-optimized RNA-dependent RNA polymerase (RdRpopt). Using this mini-replicon system, we determined that RSV NP and RdRpopt are indispensable for the eGFP expression from MR3(-)eGFP. The expression of eGFP from MR3(-)eGFP can be significantly enhanced in the presence of four viral suppressors of RNA silencing (VSRs), NSs, and P19-HcPro-γb. In addition, NSvc4, the movement protein of RSV, facilitated eGFP trafficking between cells. We also developed an antigenomic RNA3-based replicon in N. benthamiana. However, we found that the RSV NS3 coding sequence acts as a cis element to regulate viral RNA expression. Finally, we made mini-replicons representing all four RSV genomic RNAs. This is the first mini-replicon-based reverse-genetics system for monocot-infecting tenuivirus. We believe that the mini-replicon system described here will allow studies of the RSV replication, transcription, cell-to-cell movement, and host machinery underpinning RSV infection in plants. IMPORTANCE Plant-infecting segmented negative-stranded RNA (NSR) viruses are grouped into three genera: Orthotospovirus, Tenuivirus, and Emaravirus. Reverse-genetics systems have been established for members of the genera Orthotospovirus and Emaravirus. However, there is still no reverse-genetics system available for Tenuivirus. Rice stripe virus (RSV) is a monocot-infecting tenuivirus with four negative-stranded/ambisense RNA segments. It is one of the most destructive rice pathogens and causes significant damage to the rice industry in Asian countries. Due to the lack of a reliable reverse-genetics system, molecular characterizations of RSV gene functions and the host machinery underpinning RSV infection in plants are extremely difficult. To overcome this obstacle, we developed a mini-replicon-based reverse-genetics system for RSV in Nicotiana benthamiana. This is the first mini-replicon-based reverse-genetics system for tenuivirus. We consider that this system will provide researchers a new working platform to elucidate the molecular mechanisms dictating segmented tenuivirus infections in plants.

A multi-modal biosensing platform based on Ag-ZnIn2S4@Ag-Pt nanosignal probe-sensitized UiO-66 for ultra-sensitive detection of penicillin.

We designed a multi-modal biosensing platform for versatile detection of penicillin based on a unique Ag-ZnIn2S4@Ag-Pt signal probe-sensitized UiO-66 metal-organic framework. Firstly, a large number of Ag-ZnIn2S4 quantum dots (AZIS QDs) were attached to Ag-Pt NPs, preparing a new multi-signal probe AZIS QDs@Ag-Pt NPs with excellent photoelectrochemistry (PEC), electrochemiluminescence (ECL), and fluorescence (FL) signals. Moreover, the AZIS QDs@Ag-Pt NPs signal probe can well match the energy level of UiO-66 metal-organic framework (MOF) with good photoelectric property, which can reverse the PEC current of UiO-66 to reduce false positives in detection. When penicillin was present, it bound to its aptamer to release the multifunctional signal probes, which can generate PEC, ECL, and PL signals, thus realizing ultrasensitive detection of penicillin by multi-signals. This work creates a novel three-signal QDs probe, which makes a great contribution to multi-mode photoelectric sensing analysis. The LOD of this work (3.48 fg·mL-1) was much lower than the MRLs (Maximum Residue Levels) established by the EU (4 ng·mL-1). The newly developed multi-mode biosensor has good practical application values in various biological detection, food assay, and early disease diagnosis.

Dual-mode photoelectrochemical radar based on CdS quantum dot and Ce-MOF for detection of low-abundance disease-associated proteins.

Herein, we developed a convenient and versatile dual-mode electrochemiluminescence (ECL) and photoelectrochemistry (PEC) sensing radar for the detection of Prostate-specific antigen (PSA), which has important implications for detection of low-abundance disease-associated proteins. Cerium-based metal-organic framework (Ce-MOFs) were firstly modified on the electrode, showing well ECL and PEC property. In particular, a unique multifunctional Au@CdS quantum dots (QDs) probe loaded numerous QDs and antibody was fabricated, not only displaying strong ECL and PEC signals, but also having specific recognition to PSA. After the signal probe was linked to the electrode by immune reaction, much amplified signals of ECL and PEC were generated for double-mode detection of PSA. Therefore, this work proposed a multifunctional Au@CdS QDs signal probe with excellent ECL and PEC performance, and developed an ultrasensitive photoelectric biosensing platform for dual-mode detection, which provides an effective method for health monitoring of cancer patients.

[Traditional Chinese medicine syndrome factors of patients with HIV infection or AIDS in China].

OBJECTIVE: To study the characteristics of traditional Chinese medicine (TCM) syndrome factors of patients from different areas of China with human immunodeficiency virus (HIV) infection or acquired immunodeficiency syndrome (AIDS). METHODS: A cross-sectional investigation study was conducted in Henan, Guangdong and Yunnan Provinces and Xinjiang Uygur Autonomous Region of China from October 2008 to August 2010. Based on literature review and expert opinion, a clinical questionnaire of TCM syndromes was drawn up. This survey was carried out after the investigators were professionally trained. Wenfeng III Auxiliary Diagnosis and Treat System of TCM was used to analyze the frequencies of AIDS patients' signs and symptoms with scores above 70 of syndrome factors respectively. Based on this work, syndrome factors of AIDS were analyzed in different areas. RESULTS: There were 608 HIV/AIDS cases investigated from October 2008 to August 2010 in total; among them, 276 cases were from Henan, 126 cases from Guangdong, 120 cases from Xinjiang and 86 cases from Yunnan. The results of syndrome factor analysis indicated that the syndromes of four provinces were similar. HIV/AIDS patients in the four areas exhibited qi deficiency, blood deficiency, yin deficiency, yang deficiency, dampness, phlegm, qi stagnation and essence deficiency syndromes. Patients in each area also had their own characteristics, such as that the scores of dampness of Guangdong and yin deficiency of Xinjiang were higher than the other syndromes, whereas the scores of Henan Province were higher than the other areas. AIDS patients had higher scores of syndromes than HIV-infected patients. CONCLUSION: HIV/AIDS patients from different areas had similar syndrome elements. The theory of "AIDS toxin injuring primordial qi" can sum up the TCM etiology and pathogenesis of HIV/AIDS.

Myocyte-specific enhancer factor 2A is essential for zebrafish posterior somite development.

Somite development is governed tightly by genetic factors. In the large-scale mutagenesis screens of zebrafish, no mutations were linked to myocyte enhancer factor 2A (MEF2A) locus. In this study, we find that MEF2A knock-down embryos display a downward tail curvature and have U-shaped posterior somites. Furthermore, we demonstrate that MEF2A is required for Hedgehog signaling. MEF2A inhibition results in induction of apoptosis in the posterior somites. We further find that Hedgehog signaling can negatively regulate MEF2A expression in the somites. Microarray studies reveal a number of genes that are differentially expressed in the MEF2A morphants. Our studies suggest that MEF2A is essential for zebrafish posterior somite development.

Requirements of myocyte-specific enhancer factor 2A in zebrafish cardiac contractility.

Myocyte-specific enhancer factor 2A (MEF2A) regulates a broad range of fundamental cellular processes including cell division, differentiation and death. Here, we tested the hypothesis that MEF2A is required in cardiac contractility employing zebrafish as a model organism. MEF2A is highly expressed in heart as well as somites during zebrafish embryogenesis. Knock-down of MEF2A in zebrafish impaires the cardiac contractility and results in sarcomere assembly defects. Dysregulation of cardiac genes in MEF2A morphants suggests that sarcomere assembly disturbances account for the cardiac contractile deficiency. Our studies suggested that MEF2A is essential in cardiac contractility.

[Effect of external retinoic acid on Tbx1 gene during zebrafish embryogenesis].

OBJECTIVE: DiGeorge/del22q11 syndrome is one of the most common genetic causes of outflow tract and aortic arch defects in human. DiGeorge/del22q11 is thought to involve an embryonic defect restricted to the pharyngeal arches and the corresponding pharyngeal pouches. Previous studies have evidenced that retinoic acid (RA) signaling is definitely indispensable for the development of the pharyngeal arches. Tbx1, one of the T-box containing genes, is proved to be the most attractive candidate gene for DiGeorge/del22q11 syndrome. However, the interaction between RA and Tbx1 has not been fully investigated. Exploring the interaction will contribute to discover the molecular pathways disrupted in DiGeorge/del22q11 syndrome, and will also be essential for understanding genetic basis for congenital heart disease. It now seems possible that genes and molecular pathways disrupted in DiGeorge syndrome will also account for some isolated cases of congenital heart disease. Accordingly, the present study aimed to extensively study the effects of external RA on the cardiac development and Tbx1 expression during zebrafish embryogenesis. METHODS: The chemical genetics approach was applied by treating zebrafish embryos with 5 x 10(-8) mol/L RA and 10(-7) mol/L RA at 12.5 hour post fertilization (hpf). The expression patterns of Tbx1 were monitored by whole-mount in situ hybridization and quantitative real-time RT-PCR, respectively. RESULTS: The zebrafish embryos treated with 5 x 10(-8) mol/L RA and 10(-7) mol/L RA for 1.5 h at 12.5 hpf exhibited selective defects of abnormal heart tube. The results of whole-mount in situ hybridization with Tbx1 RNA probe showed that Tbx1 was expressed in cardiac region, pharyngeal arches and otic vesicle during zebrafish embryogenesis. RA treatment led to a distinct spatio-temporal expression pattern for Tbx1 from that in wild type embryo. The real-time PCR analysis showed that Tbx1 expression levels were markedly reduced by RA treatment. Tbx1 expression in the pharyngeal arches and heart were obviously down regulated compared to the wild type embryos. In contrast to 5 x 10(-8) mol/L RA-treated groups, 10(-7) mol/L RA caused a more severe effect on the Tbx1 expression level. CONCLUSION: These results suggested that there was a genetic link between RA and Tbx1 during development of zebrafish embryo. RA could produce an altered Tbx1 expression pattern in zebrafish. RA may regulate the Tbx1 expression in a dose-dependant manner. RA could represent a major epigenetic factor to cause abnormal expression of Tbx1, secondarily, disrupt the pharyngeal arch and heart development.

Wortmannin induces zebrafish cardia bifida through a mechanism independent of phosphoinositide 3-kinase and myosin light chain kinase.

Cardia bifida is an anomaly of the embryonic heart in which the bilateral myocardial rudiments fail to travel to the midline, resulting in the formation of two separate hearts in lateral positions. In zebrafish, eight loci responsible for the cardia bifida phenotype were identified in the large-scale genetic screen. Wortmannin has been reported to be a highly selective inhibitor of phosphoinositide 3-kinase and myosin light chain kinase activity. We provide the first evidence that wortmannin treatment of zebrafish embryos can induce cardia bifida in a dose-dependent manner and that wortmannin alters cardiac development between 6 and 16 h post-fertilization. In addition, we demonstrate that wortmannin induces zebrafish cardia bifida through a mechanism independent of phosphoinositide 3-kinase and myosin light chain kinase. Our findings may provide new insights into the cardiomyocyte function and disfunction.