The identification of type I MADS box genes as the upstream activators of an endosperm-specific invertase inhibitor in Arabidopsis

Background Nuclear endosperm development is a common mechanism among Angiosperms, including Arabidopsis. During nuclear development, the endosperm nuclei divide rapidly after fertilization without cytokinesis to enter the syncytial phase, which is then followed by the cellularized phase. The endosperm can be divided into three spatial domains with distinct functions: the micropylar, peripheral, and chalazal domains. Previously, we identified two putative small invertase inhibitors, InvINH1 and InvINH2, that are specifically expressed in the micropylar region of the syncytial endosperm. In addition, ectopically expressing InvINH1 in the cellularized endosperm led to a reduction in embryo growth rate. However, it is not clear what are the upstream regulators responsible for the specific expression of InvINHs in the syncytial endosperm. Results Using protoplast transient expression system, we discovered that a group of type I MADS box transcription factors can form dimers to activate InvINH1 promoter. Promoter deletion assays carried out in the protoplast system revealed the presence of an enhancer region in InvINH1 promoter, which contains several consensus cis-elements for the MADS box proteins. Using promoter deletion assay in planta, we further demonstrated that this enhancer region is required for InvINH1 expression in the syncytial endosperm. One of the MADS box genes, AGL62, is a key transcription factor required for syncytial endosperm development. Using promoter-GFP reporter assay, we demonstrated that InvINH1 and InvINH2 are not expressed in agl62 mutant seeds. Collectively, our data supports the role of AGL62 and other type I MADS box genes as the upstream activators of InvINHs expression in the syncytial endosperm. Conclusions Our findings revealed several type I MADS box genes that are responsible for activating InvINH1 in the syncytial endosperm, which in turn regulates embryo growth rate during early stage of seed development.

Deletion of the type IV secretion system promoter VirB in Brucella abortus A19 strain attenuated the virulence of the bacteria and promotes autophagy

Brucella abortus is a Gram-negative intracellular parasite bacteria causing serious health hazards in humans and animals. The type IV secretion system (T4SS), encoded by the virB promoter, has been identified as an important virulence factor for Brucella abortus, but the impact on Brucella abortus A19 remains unclear. In this study, the T4SS of Brucella abortus A19 was inactivated by deleting the virB promoter, resulting in a mutant strain A19ΔvirB. Real-time PCR and Western-blotting analysis demonstrated that T4SS-related proteins were not expressed after virB promoter deletion. Moreover, the survival rate of A19 in high salt and strong acidic environments was decreased after virB promoter deletion. Compared to the parental strain A19, the A19ΔvirB mutant strain showed reduced growth rate in TSB, decreased invasion ability to macrophages and dendritic cells, and reduced virulence of the mutant strain in macrophages, dendritic cells and mice. In addition, the A19ΔvirB mutant strain showed enhanced autophagy on macrophages and dendritic cells compared with A19, and the A19ΔvirB mutant strain was able to upregulate IL-6 and downregulate IL-10 in macrophages. These data help us to better understand the T4SS of the A19 vaccine strain and contribute to our efforts to improve Brucella vaccines.

Functional Analysis of the Lupinus luteus Cyclophilin Gene Promoter Region in Lotus japonicus

Functional analysis of promoter sequences is important to understand the regulation of gene expression. This study aimed to investigate the promoter region of the Lupinus luteus cytoplasmic cyclophilin gene (LlCyP; AF178458). After bioinformatic analysis, four promoter deletion fragments were fused to the β-glucuronidase reporter gene. We used Lotus japonicus as a model plant. After Agrobacterium rhizogenes transformation of L. japonicus, only the longest promoter region (−1055 bp to ATG) supported the β-glucuronidase expression in root nodule parenchyma. Putative cis-elements located between −1055 and −846 bp were subjected to site-directed mutagenesis. Mutations incorporated in the TGATT and AGATT motifs (cytokinin response) abolished GUS expression in nodules, but the mutated AAAGAT motif (OSE, organ-specific element) still activated the GUS expression in root nodules, mainly in cells surrounding the vascular bundle. Promoter deletion and mutation experiments suggest that cis-elements responsible for gene expression in the nodule are located in the region spanning from −1055 to −846 bp. We constructed a deletion fragment, in which the DNA sequence located between −822 and −198 bp was removed (pCYPMG). The promoter region arranged in the pCYPMG supports the expression in the parenchyma of L. japonicus nodules, but it is lower than the whole promoter region. The obtained results may be useful for transgene expression in determinate and indeterminate root nodules.

Genetic Factors Associated with Enhanced blaKPC Expression in Tn3/Tn4401 Chimeras

ABSTRACT The expression of the blaKPC gene plays a key role in carbapenem resistance in Enterobacteriaceae. However, the genetic regulators of the blaKPC gene have not been completely elucidated, especially the genes in Tn3-Tn4401 chimeras. Two novel Tn3-Tn4401 chimera isoforms were characterized in our hospital, isoform A (CTA), which harbors a 121-bp deletion containing the PX promoter and was present in 22.6% (54/239) of isolates, and isoform C (CTC), which harbors a 624-bp insertion and a P1 promoter deletion and was present in only 1 isolate. The carbapenem MICs of both isoforms were 2-fold or more higher than those of the wild type (Tn3-Tn4401 chimera, CTB), and blaKPC was most highly expressed in CTA. Bioinformatics and 5′ rapid amplification of cDNA ends (5′ RACE) experiments indicated a novel strong putative promoter, PY, at the 3′ end of the ISKpn8 gene. PY mutation nearly abrogated blaKPC expression (P < 0.01) and restored carbapenem susceptibility in all 3 isoforms. Although the mutation of PX or P1 halved blaKPC expression in CTB (P < 0.05), PX deletion caused a 68% increase in blaKPC expression (P = 0.037) in CTA. The level of blaKPC mRNA in CTC was 8-fold higher than that in InCTC, which harbors P1 (P = 0.011). These results suggest that PY is a core promoter of the blaKPC gene in the chimeras and that the deletion of the PX and P1 promoters enhanced gene expression in CTA and CTC, respectively.

Herpes Simplex Virus 2 Latency-Associated Transcript (LAT) Region Mutations Do Not Identify a Role for LAT-Associated MicroRNAs in Viral Reactivation in Guinea Pig Genital Models

ABSTRACTDespite the long-standing observation that herpes simplex virus (HSV) latency-associated transcript (LAT) promoter deletion viruses show impaired recurrence phenotypes in relevant animal models, the mechanism by which these sequences exert this phenotypic effect is unknown. We constructed and evaluated four mutant HSV-2 isolates with targeted mutations in the LAT promoter and LAT-associated microRNAs (miRNAs) affecting (i) the LAT TATA box; (ii) the LAT ICP4-binding site; (iii) miRNA I (miR-I) and miR-II (miR-I/II), which both target ICP34.5; and (iv) miR-III, which targets ICP0. While the LAT TATA box mutant caused milder acute infections than wild-type (WT) virus, there was no difference in the recurrence phenotype between these viruses. LAT and miRNA expression during latency was not impaired by this mutation, suggesting that other promoter elements may be more important for latent HSV-2 LAT expression. Mutation of the LAT ICP4-binding site also did not cause anin vivophenotypic difference between mutant and WT viruses. Acute infection and reactivation from latency of the miR-I/II mutant were similar to those of its rescuant, although the acute infection was slightly reduced in severity relative to that caused by the wild-type virus. The miR-III mutant also exhibited WT phenotypes in acute and recurrent phases of infection. While they do not rule out an effect of these elements in human latency or reactivation, these findings do not identify a specific role for LAT or LAT-associated miRNAs in the HSV-2 LAT promoter deletion phenotype in guinea pigs. Thus, other sequences in this region may play a more important role in the long-studied LAT-associated phenotype in animals.IMPORTANCEWhile it has been known for several decades that specific HSV-1 and HSV-2 sequences near the LAT promoter are required for efficient viral reactivation in animal models, the mechanism is still not known. We constructed four mutant viruses with the goal of identifying critical sequence elements and of specifically testing the hypothesis that microRNAs that are expressed during latency play a role. Determination that specific LAT promoter sequences and miRNA sequences do not influence viral reactivation of HSV-2 helps to narrow down the search for the mechanism by which the virus controls its latency and recurrence phenotype.