Rsc3K, a SMV resistance gene localized on chromosome 2 in soybean cv. Kefeng No.1, interacts with virulence determinant P3

Soybean mosaic virus (SMV) is one of the most devastating viral pathogens in Glycine max (L.) Merr (soybean). Twenty-two SMV strains (SC1-SC22) isolated in China were identified based on their responses to ten soybean cultivars. By using the F2-derived F3 (F2:3) and recombinant inbred line (RIL) populations of resistant Soybean cultivar (cv.) Kefeng No.1 × susceptible cv. Nannong 1138-2, we localized the gene mediating resistant to SMV-SC3 strain to a 90 kb interval on chromosome 2 in Kefeng No.1. Bean pod mottle vi-rus (BPMV)-induced gene silencing (VIGS) were used to study the gene function of candidate genes in the mapping interval and revealed that an recombinant gene, later named as Rsc3K, caused by internal deletion of a genomic DNA fragement in Kefeng No.1, is the resistant gene to SMV-SC3. By shuffling genes between avirulent isolate SC3 and avirulent SMV isolate 1129, we found that P3 is the virulence determinant causing resistance on Kefeng No.1. We showed the interaction between Rsc3K and P3 by the yeast two-hybrid (Y2H) and bimolecular fluorescent complementation (BiFC) assays. In conclusion, this study demonstrated that Rsc3K plays a crucial role in resistance of Kefeng No.1 to SMV-SC3 by direct interaction with viral protein P3.

Hormonal Regulatory Patterns of LaKNOXs and LaBEL1 Transcription Factors Reveal Their Potential Role in Stem Bulblet Formation in LA Hybrid Lily

In lily reproduction, the mechanism of formation of bulbs has been a hot topic. However, studies on stem bulblet formation are limited. Stem bulblets, formed in the leaf axils of under- and above-ground stems, provide lilies with a strong capacity for self-propagation. First, we showed that above-ground stem bulblets can be induced by spraying 100 mg/L 6-BA on the LA hybrid lily ‘Aladdin’, with reduced endogenous IAA and GA4 and a higher relative content of cytokinins. Then, expression patterns of three potential genes (two KNOTTED1-like homeobox (KNOX) and one partial BEL1-like homeobox (BELL)), during stem bulblet formation from our previous study, were determined by RT-qPCR, presenting a down-up trend in KNOXs and a rising tendency in BELL. The partial BELL gene was cloned by RACE from L. ‘Aladdin’ and denoted LaBEL1. Physical interactions of LaKNOX1-LaBEL1 and LaKNOX1-LaKNOX2 were confirmed by yeast two-hybrid and bimolecular fluorescence complementation assays. Furthermore, hormonal regulatory patterns of single LaKNOX1, LaKNOX2, LaBEL1, and their heterodimers, were revealed in transgenic Arabidopsis, suggesting that the massive mRNA accumulations of LaKNOX1, LaKNOX2 and LaBEL1 genes during stem bulblet formation could cause the dramatic relative increase of cytokinins and the decline of GAs and IAA. Taken together, a putative model was proposed that LaKNOX1 interacts with LaKNOX2 and LaBEL1 to regulate multiple phytohormones simultaneously for an appropriate hormonal homeostasis, which suggests their potential role in stem bulblet formation in L. ‘Aladdin’.

The Safflower bHLH Transcription Factor CtbHLH41 Negatively Regulates SA-induced Leaf Senescence Through Interaction with CtCP1

BackgroundSalicylic acid (SA) plays an important role in regulating leaf senescence. However, the molecular mechanism of leaf senescence of safflower (Carthamus tinctorius) is still elusive. In this study we found that the bHLH transcription factor (TF) CtbHLH41 in Carthamus tinctorius significantly delayed leaf senescence and inhibited the expression of senescence-related genes.ResultsIn order to explore how CtbHLH41 promotes leaf senescence, we carried out yeast two-hybrid screening. In this study, by exploring the mechanism of CtbHLH41 regulating CtCP1, it was found that CtCP1 promoted the hydrolysis of CtbHLH41 protein, accelerated the transcriptional activities of salicylic acid-mediated senescence-related genes CtSAG12 and CtSAG29, chlorophyll degradation genes CtNYC1 and CtNYE1, and accelerated leaf senescence. We found a negative SA regulator CtANS1, which interacts with CtbHLH41 and regulates its stability, thereby inhibiting CtCP1-mediated leaf senescence.ConclusionsIn short, our results provide a new insight into the mechanism of CtbHLH41 actively regulating the senescence of safflower leaves induced by SA.

Esterase D stabilizes FKBP25 to suppress mTORC1

Background Esterase D (ESD) is a nonspecific esterase that detoxifies formaldehyde. Many reports have stated that ESD activity is associated with a variety of physiological and pathological processes. However, the detailed signaling pathway of ESD remains poorly understood. Methods Considering the advantages of the small chemical molecule, our recent work demonstrated that 4-chloro-2-(5-phenyl-1-(pyridin-2-yl)-4,5-dihydro-1H-pyrazol-3-yl) phenol (FPD5) activates ESD, and will be a good tool for studying ESD further. Firstly, we determined the interaction between ESD and FK506 binding protein 25 (FKBP25) by yeast two-hybrid assay and co-immunoprecipitation (CO-IP) and analyzed the phosphorylation levels of mTORC1, P70S6K and 4EBP1 by western blot. Furthermore, we used the sulforhodamine B (SRB) and chick chorioallantoic membrane (CAM) assay to analyze cell viability in vitro and in vivo after treatment with ESD activator FPD5. Results We screened FKBP25 as a candidate protein to interact with ESD by yeast two-hybrid assay. Then we verified the interaction between ESD and endogenous FKBP25 or ectopically expressed GFP-FKBP25 by CO-IP. Moreover, the N-terminus (1–90 aa) domain of FKBP25 served as the crucial element for their interaction. More importantly, ESD reduced the K48-linked poly-ubiquitin chains of FKBP25 and thus stabilized cytoplasmic FKBP25. ESD also promoted FKBP25 to bind more mTORC1, suppressing the activity of mTORC1. In addition, ESD suppressed tumor cell growth in vitro and in vivo through autophagy. Conclusions These findings provide novel evidence for elucidating the molecular mechanism of ESD and ubiquitination of FKBP25 to regulate autophagy and cancer cell growth. The ESD/FKBP25/mTORC1 signaling pathway is involved in inhibiting tumor cell growth via regulating autophagy.

The PRY/SPRY domain of pyrin/TRIM20 interacts with β2-microglobulin to promote inflammasome formation

Pyrin/TRIM20 is expressed in the neutrophils and monocytes/macrophages and regulates caspase-1 activation and interleukin-1β maturation. Although the mutations in the PRY/SPRY domain of pyrin cause familial Mediterranean fever (FMF), the mechanism of how mutated pyrin provokes excessive inflammation in FMF patients is not well understood. The present study investigated the role of pyrin/TRIM20 in inflammation and the pathogenesis of FMF. β2-Microglobulin (β2MG) was identified as the novel pyrin ligand binding to the PRY/SPRY domain by yeast two-hybrid screenings and co-immunoprecipitation analysis. β2MG was co-localized with pyrin not only in the HEK293 cells overexpressing these proteins but also in the monosodium urate-stimulated human neutrophils in the speck-like structures. The pyrin–β2MG interaction triggered the binding of pyrin and proline–serine–threonine phosphatase interacting protein 1 (PSTPIP1) and then the subsequent recruitment of apoptosis-associated speck-like protein containing caspase recruitment domain (ASC). Caspase-1 p20 subunit, produced by pyrin inflammasome, also interacted with the pyrin PRY/SPRY domain and inhibited the pyrin–β2MG interaction. FMF-associated pyrin mutation M694V did not affect pyrin–β2MG interaction but weakened this inhibition. Our findings suggest that β2MG functions as the pyrin ligand inducing pyrin inflammasome formation and that the FMF-associated pyrin mutations weakened negative feedback of caspase-1 p20 subunit.

Resolvin D1 Suppresses H2O2-Induced Senescence in Fibroblasts by Inducing Autophagy through the miR-1299/ARG2/ARL1 Axis

ARG2 has been reported to inhibit autophagy in vascular endothelial cells and keratinocytes. However, studies of its mechanism of action, its role in skin fibroblasts, and the possibility of promoting autophagy and inhibiting cellular senescence through ARG2 inhibition are lacking. We induced cellular senescence in dermal fibroblasts by using H2O2. H2O2-induced fibroblast senescence was inhibited upon ARG2 knockdown and promoted upon ARG2 overexpression. The microRNA miR-1299 suppressed ARG2 expression, thereby inhibiting fibroblast senescence, and miR-1299 inhibitors promoted dermal fibroblast senescence by upregulating ARG2. Using yeast two-hybrid assay, we found that ARG2 binds to ARL1. ARL1 knockdown inhibited autophagy and ARL1 overexpression promoted it. Resolvin D1 (RvD1) suppressed ARG2 expression and cellular senescence. These data indicate that ARG2 stimulates dermal fibroblast cell senescence by inhibiting autophagy after interacting with ARL1. In addition, RvD1 appears to promote autophagy and inhibit dermal fibroblast senescence by inhibiting ARG2 expression. Taken together, the miR-1299/ARG2/ARL1 axis emerges as a novel mechanism of the ARG2-induced inhibition of autophagy. Furthermore, these results indicate that miR-1299 and pro-resolving lipids, including RvD1, are likely involved in inhibiting cellular senescence by inducing autophagy.

Yeast Two-Hybrid Screen Identifies PKA-Riα Interacting Proteins during Mouse Spermiogenesis

cAMP-dependent protein kinase (PKA) signaling plays various roles during mammalian spermatogenesis, ranging from the regulation of gene expression to the modulation of sperm motility. However, the molecular mechanisms that govern the multifaceted functions of PKA during spermatogenesis remain largely unclear. We previously found that PKA regulatory subunit I α (RIα) and catalytic subunit α (Cα) co-sediment with polyribosomal fractions of mouse testis lysate on sucrose gradient and the stimulation of PKA activity facilitates protein synthesis in post-meiotic elongating spermatids, indicating that type I PKA is intricately associated with protein translation machinery and regulates protein synthesis during mouse spermiogenesis. Since PKA activity is often regulated by interacting proteins that form complexes with its regulatory subunits, the identification of PKA-RIα interacting proteins in post-meiotic spermatogenic cells will facilitate our understanding of its regulatory roles in protein synthesis and spermiogenesis. In the present study, we applied a yeast two-hybrid screen to identify PKA-Riα-binding proteins using a cDNA library generated from mouse round and elongating spermatids. Numerous proteins were found to potentially interact with PKA-RIα, including proteostasis modulators, metabolic enzymes, cytoskeletal regulators, and mitochondrial proteins, many of which are specifically expressed in testes. Consistently, the examination of MENA (mouse ENA/VASP homolog) in developing mouse testes suggested that post-meiotic spermatogenic cells express a short isoform of MENA that interacts with PKA-RIα in yeast two-hybrid assay. The identification of PKA-RIα interacting proteins provides us solid basis to further explore how PKA signaling regulates protein synthesis and cellular morphogenesis during mouse spermatogenesis.

JAZ8 Interacts With VirE3 Attenuating Agrobacterium Mediated Root Tumorigenesis

Agrobacterium tumefaciens can cause crown gall tumors by transferring both an oncogenic piece of DNA (T-DNA) and several effector proteins into a wide range of host plants. For the translocated effector VirE3 multiple functions have been reported. It acts as a transcription factor in the nucleus binding to the Arabidopsis thaliana pBrp TFIIB-like protein to activate the expression of VBF, an F-box protein involved in degradation of the VirE2 and VIP1 proteins, facilitating Agrobacterium-mediated transformation. Also VirE3 has been found at the plasma membrane, where it could interact with VirE2. Here, we identified AtJAZ8 in a yeast two-hybrid screening with VirE3 as a bait and confirmed the interaction by pull-down and bimolecular fluorescence complementation assays. We also found that the deletion of virE3 reduced Agrobacterium virulence in a root tumor assay. Overexpression of virE3 in Arabidopsis enhanced tumorigenesis, whereas overexpression of AtJAZ8 in Arabidopsis significantly decreased the numbers of tumors formed. Further experiments demonstrated that AtJAZ8 inhibited the activity of VirE3 as a plant transcriptional regulator, and overexpression of AtJAZ8 in Arabidopsis activated AtPR1 gene expression while it repressed the expression of AtPDF1.2. Conversely, overexpression of virE3 in Arabidopsis suppressed the expression of AtPR1 whereas activated the expression of AtPDF1.2. Our results proposed a novel mechanism of counter defense signaling pathways used by Agrobacterium, suggesting that VirE3 and JAZ8 may antagonistically modulate the salicylic acid/jasmonic acid (SA/JA)-mediated plant defense signaling response during Agrobacterium infection.