Analysis of bacterial and fungal community structure in replant strawberry rhizosphere soil with denaturing gradient gel electrophoresis

High quality DNA is the basis of analyzing bacterial and fungal community structure in replant strawberry rhizosphere soil with the method of denaturing gradient gel electrophoresis (DGGE). DNA of soil  microorganisms was extracted from the rhizosphere soil of strawberries planted in different replanted  years (0, two, six and seven), respectively, and crude DNA was purified after extraction. Three methods  were established to evaluate the effects of cetyl trimethylammonium bromide (CTAB),  polyvinylpolypyrolidone (PVPP), proteinase K and bacteriolytic enzymes on DNA extraction. DNA  fragments above 23 kb in size were isolated well by method 1 (1% CTAB, proteinase K, no PVPP, no  bacteriolytic enzyme) and method 3 (no CTAB, no proteinase K, 3% PVPP, bacteriolytic enzyme). Method 3 got the best yields 43.06 ìg/g, and A260/A280 and A260/A230 were 1.1623 and 0.8135, respectively,  which could ensure the veracity of subsequent DGGE analysis. Method 2 (3% CTAB, no proteinase K, no PVPP, no bacteriolytic enzyme) could not extract enough DNA to do the next PCR-DGGE analysis.  F341/R534 and FR1/FF390 primers were used to amplify the 16S rDNA V3 region of bacteria and 18S rDNA of fungi, and the expected fragments of 230 bp 16S rDNA V3 region and 390 bp 18S rDNA were amplified. The results of DGGE analysis showed that there were common and specific bacterial and fungal  communities in different replant soils of strawberry. There were 84 and 54% similarity of bacterial and  fungal communities between different replant soils. The numbers of both bacterial and fungal communities increased in the replant strawberry soil, they were positively correlated with the replant years. As the  number of replant years increased from two to seven years, while the ratio of bacteria/fungi was  decreased from 2.29 to 1.46 in the rhizosphere soils planted with strawberries.Key words: Rhizosphere soil, bacterial community, fungal community, replant strawberry, fruiting fields

Arabidopsis AAR2, a conserved splicing factor in eukaryotes, acts in microRNA biogenesis

MicroRNAs (miRNAs) play an essential role in plant growth and development, and as such, their biogenesis is fine-tuned via regulation of the core microprocessor components. Here, we report that Arabidopsis AAR2, a homolog of a U5 snRNP assembly factor in yeast and humans, not only acts in splicing but also promotes miRNA biogenesis. AAR2 interacts with the microprocessor component hyponastic leaves 1 (HYL1) in the cytoplasm, nucleus, and dicing bodies. In aar2 mutants, abundance of nonphosphorylated HYL1, the active form of HYL1, and the number of HYL1-labeled dicing bodies are reduced. Primary miRNA (pri-miRNA) accumulation is compromised despite normal promoter activities of MIR genes in aar2 mutants. RNA decay assays show that the aar2-1 mutation leads to faster degradation of pri-miRNAs in a HYL1-dependent manner, which reveals a previously unknown and negative role of HYL1 in miRNA biogenesis. Taken together, our findings reveal a dual role of AAR2 in miRNA biogenesis and pre-messenger RNA splicing.Fil: Fan, Lusheng. University of California; Estados UnidosFil: Gao, Bin. University of California; Estados UnidosFil: Xu, Ye. University of California; Estados UnidosFil: Flynn, Nora. University of California; Estados UnidosFil: Le, Brandon. University of California; Estados UnidosFil: You, Chenjiang. Fudan University; ChinaFil: Li, Shaofang. Fudan University; ChinaFil: Achkar, Natalia Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Manavella, Pablo Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Yang, Zhenbiao. University of California; Estados UnidosFil: Chen, Xuemei. University of California; Estados Unido

Arabidopsis blue light receptor phototropin 1 undergoes blue light-induced activation in membrane microdomains

Phototropin (phot)-mediated signaling initiated by blue light (BL) plays a critical role in optimizing photosynthetic light capture at the plasma membrane (PM) in plants. However, the mechanisms underlying the regulation of phot activity at the PM in response to BL remain largely unclear. In this study, by single-particle tracking and step-wise photobleaching analysis we demonstrated that in the dark phot1-GFP proteins remain in an inactive state and mostly present as a monomer. The phot1-GFP diffusion rate and its dimerization increased in a dose-dependent manner in response to BL. In contrast, BL did not affect the lateral diffusion of kinase-inactive phot1 -GFP, whereas it did enhance its dimerization, suggesting that phot1 dimerization is independent of its phosphorylation. Förster resonance energy transfer-fluorescence lifetime imaging microscopy (FRET-FLIM) analysis revealed that the interaction between phot1-GFP and AtRem1.3-mCherry was enhanced along with increased time of BL treatment. However, the BL-dependent interaction was not obvious in plants co-expressing phot1 -GFP and AtRem1.3-mCherry, implicating that BL facilitated the translocation of functional phot1-GFP into AtRem1.3-labeled microdomains to activate phot-mediated signaling. Conversely, sterol depletion attenuated phot1-GFP dynamics, dimerization, and phosphorylation. Taken together, these results indicate that membrane microdomains act as an organizing platform essential for proper function of activated phot1 at the PM

HPV16 E6/E7 -based mRNA vaccine is therapeutic in mice bearing aggressive HPV-positive lesions

HPV (Human papillomavirus) affects 600,000 people worldwide each year. Almost all cervical cancers are associated with a past HPV infection. In particular, the positivity to the high-risk type HPV16 is detected in most of the invasive cervical cancers. FDA has approved prophylactic vaccines that protect against new HPV16 infections, but do not induce immunity in those patients with established infections or neoplasms. To date, no therapeutic vaccine targeting HPV16-associated lesions has been authorized. We have developed an mRNA-based vaccine against the HPV16 late oncoproteins E6 and E7, which are abundantly and exclusively expressed in high-grade squamous intraepithelial lesions (HSILs), a stage of the cervical disease that precedes the progression to carcinoma. Our in vitro and in vivo studies demonstrated that the translated mRNA is functional and elicits an antigen-specific adaptive immune response. Upon immunization with the vaccine, mice with HPV16+ lesions exhibited tumor growth inhibition, extension of lifespan, and development of a protective immune memory. In light of these results and the remarkable clinical success of mRNA vaccines against SARS-CoV2, we believe that our mRNA-based therapeutic vaccine has the potential to offer a non-invasive treatment alternative to the current standard of care for HPV16+ HSILs

Randomized Algorithms for Motif Detection

Abstract. Motivation: Motif detection for DNA sequences has many important applications in biological studies, e.g., locating binding sites and regulatory signals, and designing genetic probes etc. In this paper, we propose a randomized algorithm, design an improved EM algorithm and combine them to form a software. Results: (1) We design a randomized algorithm for consensus pattern problem. We can show that with high probability, our randomized algorithm finds a pattern in polynomial time with cost error at most ɛ × l for each string, where l is the length of the motif and ɛ can be any positive number given by the user. (2) We design an improved EM (Expectation Maximization) algorithm that outperforms the original EM algorithm. (3) We develop a software MotifDetector that uses our randomized algorithm to find good seeds and uses the improved EM algorithm to do local search. We compare MotifDetector with Buhler and Tompa’s PROJEC-TION which is considered to be the best known software for motif detection. Simulations show that MotifDetector is slower than PROJECTION when the pattern length is relatively small, and outperforms PROJEC-TION when the pattern length becomes large. Availability: Free fro

Endocytosis and its regulation in plants

Endocytosis provides a major route of entry for membrane proteins, lipids, and extracellular molecules into the cell. Recent evidence indicates that multiple cellular processes require endocytosis, including nutrient uptake, signaling transduction, and plant-microbe interactions. Also, advanced microscopy, combined with biochemical and genetic approaches, has provided more insights into the molecular machinery and functions of endocytosis in plants. Here we review mechanisms of the clathrin-dependent and membrane microdomain-associated endocytic routes in plant cells. In addition, degradation of endocytosed proteins and endosomal sorting complex required for transport (ESCRT)-mediated vesicle formation at the endosome are discussed. Finally, we summarize the essential roles of various regulators during plant endocytosis