HPV16 E7 protein antagonizes TNF-α-induced apoptosis of cervical cancer cells via Daxx/JNK pathway.

Human papillomavirus (HPV) E7 protein as an important viral factor was involved in the progression of cervical cancer by mediating the cellular signaling pathways. Daxx (Death domain-associated protein) can interact with a variety of proteins to affect the viral infection process. However, the interaction and its related function between HPV16 E7 and Daxx have not been adequately investigated. Here, it was found that HPV16 E7 can interact with Daxx in HeLa or C33A cells by co-immunoprecipitation. HPV16 E7 protein treatment can up-regulate Daxx protein expression, while the interference in Daxx expression and the agonists for JNK can both reduce the antagonistic effects of HPV16 E7 on TNF-α-induced apoptosis, suggesting that Daxx/JNK pathway may be involved in the anti-apoptotic activity of HPV16 E7.

OsHSD2 interaction with and phosphorylation by OsCPK21 is essential for lipid metabolism during rice caryopsis development.

Rice calcium-dependent protein kinase 21 (OsCPK21) is specifically and highly expressed throughout reproductive development and plays a critical role in rice pollen development by indirectly regulating the MIKC*-type MADS box transcription factor. However, little is known about the function of OsCPK21 in rice caryopsis development. In this study, we performed an in vitro pull-down experiment followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and identified hydroxysteroid dehydrogenase 2 (HSD2) as a candidate OsCPK21-interacting protein in 25 DAF (days after flowering) rice caryopses. Then, we verified the interaction between OsCPK21 and OsHSD2 using yeast two-hybrid and bimolecular fluorescence assays and revealed the in vitro phosphorylation of OsHSD2 by OsCPK21. Furthermore, oscpk21 and oshsd2 mutants were generated by the CRISPR/Cas9 technique, and we found that the lipid profiles were drastically changed in both oscpk21 and oshsd2, implying that OsHSD2 phosphorylated by OsCPK21 regulates lipid abundance in caryopsis development, thereby providing a potential target for the genetic improvement of rice grain quality in future lipid-related breeding and biotechnology applications.

Transcriptomic identification of long noncoding RNAs and their hormone-associated nearby coding genes involved in the differential development of caryopses localized on different branches in rice.

Long noncoding RNAs (lncRNAs) play important regulatory roles in caryopsis development and grain size in rice. However, whether there exist differences in lncRNA expression between caryopses located on primary branches (CPB) and caryopses located on secondary branches (CSB) that contribute to their differential development remains elusive. Here, we performed transcriptome-wide analysis to identify 2,273 lncRNAs expressed in CPB and CSB at 0, 5, 12, and 20 days after flowering (DAF). Although these lncRNAs were widely distributed, the majority were located in intergenic regions of the 12 rice chromosomes. Based on gene expression cluster analysis, lncRNAs expressed in CPB and CSB were clustered into two subtypes in a position-independent manner: one includes 0- and 5-DAF CPB and CSB, and 12-DAF CSB; the second includes 12-DAF CPB and 20-DAF CPB and CSB. Furthermore, according to the expression value of each lncRNA, K-means cluster analysis revealed 135 early-stage, 116 middle-stage, and 114 late-stage expression-delayed lncRNAs in CSB. Then, we analyzed the expression values of the expression-delayed lncRNAs and nearby coding genes (100 kb upstream and downstream of the lncRNAs), and found 631 lncRNA-mRNA pairs, including 258 lncRNAs and 571 nearby coding genes, some of which are related to hormone-regulated grain development. These results suggested that expression-delayed lncRNAs in CSB may regulate the development of CPB and CSB, providing insight into the mechanism underlying the developmental differences between CPB and CSB, and the differences in grain yield.

Transcriptomic analysis reveals the contribution of auxin on the differentially developed caryopses on primary and secondary branches in rice.

Generally, the caryopses located on proximal secondary branches (CSB) have smaller grain size and slower and poorer filling rate than those on apical primary branches (CPB) in rice, which greatly limits the grain yield potential fulfillment. However, the key regulators determining the developmental differences between CPB and CSB remain elusive. Here, we have performed transcriptomic analysis in CPB and CSB at four developmental stages [0, 5, 12 and 20 days after fertilization (DAF)] using high-throughput RNA-sequencing technique. Based on gene expression cluster analysis, the genes expressed in CPB and CSB were clustered into two subtypes in a positional-independent manner: one includes 0- and 5-DAF CPB and CSB, and 12-DAF CSB; another includes 12-DAF CPB, 20-DAF CPB and CSB. Moreover, according to the expression value of each gene, K-mean cluster analysis showed that the K4 to K6 classifiers contain the genes highly expressed in 5-DAF CPB and 12-DAF CSB, which were enriched in DNA synthesis, protein synthesis and cell proliferation mainly responsible for grain size decision. Then, functional enrichment analysis in Gene Ontology database showed that auxin-related genes were relatively enriched, indicating that auxin might be the key determinant for gene expression in K4 to K6 classifiers. Finally, the application of exogenous IAA in CSB before fertilization promoted gene expression, caryopsis development and grain weight closer to that in CPB, providing a molecular framework to optimize CSB development and potential targets for increasing grain yield.

Auxin apical dominance governed by the OsAsp1-OsTIF1 complex determines distinctive rice caryopses development on different branches.

In rice (Oryza sativa), caryopses located on proximal secondary branches (CSBs) have smaller grain size and poorer grain filling than those located on apical primary branches (CPBs), greatly limiting grain yield. However, the molecular mechanism responsible for developmental differences between CPBs and CSBs remains elusive. In this transcriptome-wide expression study, we identified the gene Aspartic Protease 1 (OsAsp1), which reaches an earlier and higher transcriptional peak in CPBs than in CSBs after pollination. Disruption of OsAsp1 expression in the heterozygous T-DNA line asp1-1+/-eliminated developmental differences between CPBs and CSBs. OsAsp1 negatively regulated the transcriptional inhibitor of auxin biosynthesis, OsTAA1 transcriptional inhibition factor 1 (OsTIF1), to preserve indole-3-acetic acid (IAA) apical dominance in CPBs and CSBs. IAA also facilitated OsTIF1 translocation from the endoplasmic reticulum (ER) to the nucleus by releasing the interaction of OsTIF1 with OsAsp1 to regulate caryopses IAA levels via a feedback loop. IAA promoted transcription of OsAsp1 through MADS29 to maintain an OsAsp1 differential between CPBs and CSBs during pollination. Together, these findings provide a mechanistic explanation for the distributed auxin differential between CPBs and CSBs to regulate distinct caryopses development in different rice branches and potential targets for engineering yield improvement in crops.

Calcium-dependent protein kinase 21 phosphorylates 14-3-3 proteins in response to ABA signaling and salt stress in rice.

The calcium-dependent protein kinases (CDPKs) are a class of plant-specific kinase that directly bind Ca2+ and mediate the calcium-signaling pathways to play important physiological roles in growth and development. The rice genome contains 31 CDPK genes, one of which, OsCPK21, is known to modulate the abscisic acid (ABA) and salt stress responses in this crop; however, the molecular mechanisms underlying this regulation are largely unknown. In the present study, we performed yeast two-hybrid screening, glutathione S-transferase pull-down, co-immunoprecipitation, and bimolecular fluorescence complementation assays to confirm the interaction between OsCPK21 and one of its putative targets, Os14-3-3 (OsGF14e). We used an in vitro kinase assay and site-directed mutagenesis to verify that OsCPK21 phosphorylates OsGF14e at Tyr-138. We used real-time PCR to reveal that several ABA and salt inducible genes were more highly expressed in the OsCPK21-OE and OsGF14e WT-OE plants than in the mutant OsGF14e Y138A-OE and wild-type plants. These results suggest that OsCPK21 phosphorylates OsGF14e to facilitate the response to ABA and salt stress.

FRET-based glucose imaging identifies glucose signalling in response to biotic and abiotic stresses in rice roots.

Glucose is the primary energy provider and the most important sugar-signalling molecule, regulating metabolites and modulating gene expression from unicellular yeast to multicellular plants and animals. Therefore, monitoring intracellular glucose levels temporally and spatially in living cells is an essential step for decoding the glucose signalling in response to biotic and abiotic stresses. In this study, the genetically encoded FRET (Förster resonance energy transfer) nanosensors, FLIPglu-2µ∆13 and FLIPglu-600µΔ13, were used to measure cytosolic glucose dynamics in rice plants. First, we found that the FRET signal decreased in response to external glucose in a concentration-dependent manner. The glucose concentration at which the cytosolic level corresponded to the K0.5 value for FLIPglu-2µΔ13 was approximately 10.05µM, and that for FLIPglu-600µΔ13 was 0.9mM, respectively. The substrate selectivity of nanosensors for glucose and its analogues is D-Glucose>2-deoxyglucose>3-O-methylglucose>L-Glucose. We further showed that the biotic elicitors (flg22 and chitin) and the abiotic elicitors (osmotic stress, salinity and extreme temperature) induce the intracellular glucose increases in the detached root segments of transgenic rice containing FLIPglu-2µΔ13 in a stimulus-specific manner, but not in FLIPglu-600µΔ13 transgenic lines. These results demonstrated that FRET nanosensors can be used to detect increases in intracellular glucose within the physiological range of 0.2-20µM in response to various stimuli in transgenic rice root cells, which indicated that intracellular glucose may act as a potential secondary messenger to connect extracellular stimuli with cellular physiological responses in plants.