Calreticulin expression and localization in relation to exchangeable Ca2+ during pollen development in Petunia

Background Pollen development in the anther in angiosperms depends on complicated cellular interactions associated with the expression of gametophytic and sporophytic genes which control fundamental processes during microsporo/gametogenesis, such as exo/endocytosis, intracellular transport, cell signaling, chromatin remodeling, and cell division. Most if not all of these cellular processes depend of local concentration of calcium ions (Ca2+). Work from our laboratory and others provide evidence that calreticulin (CRT), a prominent Ca2+-binding/buffering protein in the endoplasmic reticulum (ER) of eukaryotic cells, may be involved in pollen formation and function. Here, we show for the first time the expression pattern of the PhCRT1 gene and CRT accumulation in relation to exchangeable Ca2+ in Petunia hybrida developing anther, and discuss probable roles for this protein in the male gametophyte development. Results Using northern hybridization, western blot analysis, fluorescent in situ hybridization (FISH), immunocytochemistry, and potassium antimonate precipitation, we report that PhCRT1 is highly expressed in the anther and localization pattern of the CRT protein correlates with loosely bound (exchangeable) Ca2+ during the successive stages of microsporo/gametogenesis. We confirmed a permanent presence of both CRT and exchangeable Ca2+ in the germ line and tapetal cells, where these factors preferentially localized to the ER which is known to be the most effective intracellular Ca2+ store in eukaryotic cells. In addition, our immunoblots revealed a gradual increase in CRT level from the microsporocyte stage through the meiosis and the highest CRT level at the microspore stage, when both microspores and tapetal cells show extremely high secretory activity correlated with the biogenesis of the sporoderm. Conclusion Our present data provide support for a key role of CRT in developing anther of angiosperms – regulation of Ca2+ homeostasis during pollen grains formation. This Ca2+-buffering chaperone seems to be essential for pollen development and maturation since a high rate of protein synthesis and protein folding within the ER as well as intracellular Ca2+ homeostasis are strictly required during the multi-step process of pollen development.

Transcriptomic analysis of ribosome biogenesis and pre-rRNA processing during growth stress in Entamoeba histolytica

Ribosome biogenesis, a multi-step process involving the transcription, modification, folding and processing of rRNA is the major consumer of cellular energy. It involves the sequential assembly of ribosomal proteins (RP)s via more than 200 ribogenesis factors. Unlike model organisms where transcription of rRNA and RP genes slows down during stress, in Entamoeba histolytica, pre-rRNA synthesis continues, and unprocessed pre-rRNA accumulates. To gain insight into the vast repertoire of ribosome biogenesis factors and understand the major components playing role during stress we computationally identified the ribosome biogenesis factors in E. histolytica. Of the total ~279 S. cerevisiae proteins, we could only find 188 proteins in E. histolytica. Some of the proteins missing in E. histolytica were also missing in humans. A number of proteins represented by multiple genes in S. cerevisiae had only a single copy in E. histolytica. It was interesting to note that E. histolytica lacked mitochondrial ribosome biogenesis factors and had far less RNase components as compared to S. cerevisiae. Northern hybridization using probes from different spacer regions depicted the accumulation of unprocessed intermediates during stress. Transcriptomic studies revealed the differential regulation of a number of ribosomal factors both in serum-starved and RRP6KD conditions. The ARB1 protein involved at multiple steps of ribosome biogenesis and NEP1 and TSR3 involved in chemical modification of 18S rRNA previously shown to accumulate pre-rRNA precursors upon downregulation in S. cerevisiae and humans were included. The data reveals the importance of some of the major factors required for regulating pre-rRNA processing during stress. This is the first report on the complete repertoire of ribosome biogenesis factors in E. histolytica.

Global analysis of the RpaB regulon based on the positional distribution of HLR1 sequences and comparative differential RNA-Seq data

ABSTRACTThe transcription factor RpaB regulates the expression of genes encoding photosynthesis-associated proteins during light acclimation. The binding site of RpaB is the HLR1 motif, a pair of imperfect octameric direct repeats, separated by two random nucleotides. Here, we used high-resolution mapping data of transcriptional start sites (TSSs) in the modelSynechocystissp. PCC 6803 in conjunction with the positional distribution of HLR1 sites for the global prediction of the RpaB regulon. The results demonstrate that RpaB regulates the expression of more than 150 promoters, driving the transcription of protein-coding and non-coding genes and antisense transcripts under low light and upon the shift to high light when DNA binding activity is lost. Transcriptional activation by RpaB is achieved when the HLR1 motif is located 66 to 45 nt upstream, repression occurs when it is close to or overlapping the TSS. Selected examples were validated by multiple experimental approaches, including chromatin affinity purification, reporter gene, northern hybridization and electrophoretic mobility shift assays. We found that RpaB controlsssr2016/pgr5, which is involved in cyclic electron flow and state transitions; six out of nine ferredoxins; three of four FtsH proteases;gcvP/slr0293, encoding a crucial photorespiratory protein; andnirAandisiAfor which we suggest cross-regulation with the transcription factors NtcA or FurA, respectively. In addition to photosynthetic gene functions, RpaB contributes to the control of genes affiliated with nitrogen assimilation, cofactor biosyntheses, the CRISPR system and the circadian clock, making it one of the most versatile regulators in cyanobacteria.Significance StatementRpaB is a transcription factor in cyanobacteria and in the chloroplasts of several lineages of eukaryotic algae. Like other important transcription factors, the gene encoding RpaB cannot be deleted, making the study of deletion mutants impossible. Based on a bioinformatic approach, we increased the number of known genes controlled by RpaB by a factor of 5. Depending on the distance to the TSS, RpaB mediates transcriptional activation or repression. The high number and functional diversity among its target genes and co-regulation with other transcriptional regulators characterize RpaB as a regulatory hub.

Expression of β-defensin gene in potato confers enhanced resistance to Ralstonia Solanacearum L.

An optimized methodology of <em>Agrobacterium</em>-mediated stable genetic transformation of potato (<em>Solanum tuberosum </em>L.) using the shoot organogenesis potential of internodal stem segments for increased resistance to bacterial plant pathogen, <em>Ralstonia solanacearum</em> L. was developed. Improvised plant regeneration protocol for expression of antimicrobial β-defensin transgene and efficient selection of tissues in plant selectable marker, kanamycin sulphate was successfully utilized for transformation of potato. Stable integration and expression of antimicrobial peptide was observed in plant tissues and validated by associated molecular analysis by RT PCR, Southern hybridization, northern hybridization and western blotting of the infected tissues. The bacterial wilt disease progression was monitored in controlled greenhouse and Percent Disease Index (PDI) was measured by analysis of variance (ANOVA) that selected superior resistant plants. These transformed plants were able to contain the disease progression and complete the life cycle stages and developed healthy tubers.

Dark Side of Molecular Techniques: How to Fix It ?

Many fundamental molecular techniques (PCR, Microarray, Southern and northern hybridization, siRNA, CRISPR/Cas9 etc.) developed so far shows errors. I wish to highlight these molecular techniques are developed on basis of Watson-Crick DNA model, ignoring the concept of parallel stranded DNA. Through this opinion article, I wish to highlight specificity and accuracy of these molecular techniques can be enhanced by considering both parallel and anti parallel hybridization of DNA. Hopefully my views will also solve issue of irreproducibility in life science research.

IDENTIFICATION OF CDNA FRAGMENT TIGHTLY LINKED TO NV AND TM-2 LOCI IN TOMATO

Tm-2 is a resistance gene in tomato to Tomato Mosaic Virus (ToMV), located in heterochromatic region of chromosome nine. Since map based cloning difficult to perform for identify the gene on that region, we apply differential display approach by using two near-isogenic tomato lines (NILs), one without Tm-2 and the other with Tm-2 to identify cDNAs of the transcripts from the region surrounding the Tm-2 locus. Among the 150 combinations of three anchor primers and fifty arbitrary primers, 10 combinations generated cDNA polymorphic bands. Out of them, only one combination of CA6, exhibited polymorphic band under southern blot analysis, subsequently a genetic experiment showed that the CA6 locus tightly linked to the Tm-2 locus. The CA6 fragment also hybridized to genomic DNA fragments from a tomato line carrying Tm-2a, a line of L. peruvianum from which Tm-2a originated, and a tomato line carrying another Tm-2-like gene. A northern hybridization blotting result suggested that the gene corresponding to CA6 fragment was constitutively transcribed.

Mutational Analysis of the Antitoxin in the Lactococcal Type III Toxin-Antitoxin System AbiQ

ABSTRACTThe lactococcal abortive phage infection mechanism AbiQ recently was classified as a type III toxin-antitoxin system in which the toxic protein (ABIQ) is regulated following cleavage of its repeated noncoding RNA antitoxin (antiQ). In this study, we investigated the role of the antitoxin in antiphage activity. The cleavage ofantiQby ABIQ was characterized using 5′ rapid amplification of cDNA ends PCR and was located in an adenine-rich region ofantiQ. We next generated a series of derivatives with point mutations withinantiQor with various numbers ofantiQrepetitions. These modifications were analyzed for their effect on the antiphage activity (efficiency of plaquing) and on the endoribonuclease activity (Northern hybridization). We observed that increasing or reducing the number ofantiQrepeats significantly decreased the antiphage activity of the system. Several point mutations had a similar effect on the antiphage activity and were associated with changes in the digestion profile ofantiQ. Interestingly, a point mutation in the putative pseudoknot structure ofantiQmutants led to an increased AbiQ antiphage activity, thereby offering a novel way to increase the activity of an abortive infection mechanism.