OsMADS6 Controls Flower Development by Activating Rice FACTOR OF DNA METHYLATION LIKE1.

OsMADS6, an ancient AGAMOUS-LIKE6 (AGL6)-like gene, has essential functions in specifying floral organ and meristem identity in rice (Oryza sativa). However, how AGL6 genes control flower development remains largely unknown. In this study, we report that OsMADS6 directly targets FACTOR OF DNA METHYLATION LIKE 1 (OsFDML1), a rice homolog of the SUPPRESSOR OF GENE SILENCING3-like gene FACTOR OF DNA METHYLATION 1 (FDM1) from Arabidopsis (Arabidopsis thaliana). Arabidopsis FDM1 is involved in RNA-directed DNA methylation and OsFDML1 regulates flower development. The expression of OsFDML1 overlaps with that of OsMADS6 in the palea primordia and the ovule, and OsMADS6 directly promotes OsFDML1 expression through binding to regions containing putative CArG motifs within the OsFDML1 promoter during rice spikelet development. Consistent with the phenotypes of osmads6 mutants, the osfdml1 mutants showed floral defects, including altered palea identity with lemma-like shape containing no marginal region of palea, increased numbers of stigmas and fused carpels, and meristem indeterminacy. Moreover, transgenic plants overexpressing OsFDML1 displayed floral defects, such as abnormal paleae. Phylogenetic analysis showed that OsFDML1 homologs exist only in terrestrial plants. In addition, protein-protein interaction assays showed that OsFDML1 interacts with its close paralog OsFDML2, similar to the activity of OsFDML1 homologs in Arabidopsis. These results provide insight into how the ancient AGL6 gene regulates floral development.

Interactions between FLORAL ORGAN NUMBER4 and floral homeotic genes in regulating rice flower development.

The floral meristem (FM) is self-maintaining at the early stages of flower development, but it is terminated when a fixed number of floral organs are produced. The FLORAL ORGAN NUMBER4 (FON4; also known as FON2) gene, an ortholog of Arabidopsis CLAVATA3 (CLV3), is required for regulating FM size and determinacy in rice. However, its interactions with floral homeotic genes remain unknown. Here, we report the genetic interactions between FON4 and floral homeotic genes OsMADS15 (an A-class gene), OsMADS16 (also called SUPERWOMAN1, SPW1, a B-class gene), OsMADS3 and OsMADS58 (C-class genes), OsMADS13 (a D-class gene), and OsMADS1 (an E-class gene) during flower development. We observed an additive phenotype in the fon4 double mutant with the OsMADS15 mutant allele dep (degenerative palea). The effect on the organ number of whorl 2 was enhanced in fon4 spw1. Double mutant combinations of fon4 with osmads3, osmads58, osmads13, and osmads1 displayed enhanced defects in FM determinacy and identity, respectively, indicating that FON4 and these genes synergistically control FM activity. In addition, the expression patterns of all the genes besides OsMADS13 had no obvious change in the fon4 mutant. This work reveals how the meristem maintenance gene FON4 genetically interacts with C, D, and E floral homeotic genes in specifying FM activity in monocot rice.

A histidine kinase PmHHK1 regulates polar growth, sporulation and cell wall composition in the dimorphic fungus Penicillium marneffei.

Penicillium marneffei is an important opportunistic dimorphic fungal pathogen that can cause fatal systemic mycosis in AIDS patients. To find new ways of overcoming infection, candidate virulence associated genes and virulence mechanisms are under intensive investigation. In the present study, we have examined the function of a novel P. marneffei histidine kinase gene (PmHHK1) using dsRNAi mediated by Agrobacterium tumefaciens. Our results showed that reduction of PmHHK1 expression produces significant changes in morphogenesis (including polarized growth), sporulation and cell wall composition. Two-component signaling systems are widespread in the eukaryotes outside the animal kingdom, and could be potential drug targets for antifungal chemotherapy.

Depression of biofilm formation and antibiotic resistance by sarA disruption in Staphylococcus epidermidis.

AIM: To study the effects of disruption of sarA gene on biofilm formation and antibiotic resistance of Staphylococcus epidermidis (S. epidermidis). METHODS: In order to disrupt sarA gene, the double-crossover homologous recombination was applied in S. epidermidis RP62A, and tetracycline resistance gene (tet) was used as the selective marker which was amplified by PCR from the pBR322 and inserted into the locus between sarA upstream and downstream, resulting in pBT2delta sarA. By electroporation, the plasmid pBT2delta sarA was transformed into S. epidermidis. Gene transcription was detected by real-time reverse transcription-PCR (RT-PCR). Determination of biofilm was performed in 96-well flat-bottomed culture plates, and antibiotic resistance was analyzed with test tube culture by spectrophotometry at 570 nm respectively. RESULTS: A sarA disrupted strain named S. epidermidis RP62Adelta sarA was constructed, which was completely defective in biofilm formation, while the sarA complement strain RP62Adelta sarA (pHPS9sarA) restored the biofilm formation phenotype. Additionally, the knockout of sarA resulted in decreased erythromycin and kanamycin resistance of S. epidermidis RP62A. Compared to the original strain, S. epidermidis RP62Adelta sarA had an increase of the sensitivity to erythromycin at 200-400 microg/mL and kanamycin at 200-800 microg/mL respectively. CONCLUSION: The knockout of sarA can result in the defect in biofilm formation and the decreased erythromycin and kanamycin resistance in S. epidermidis RP62A.

Integration of E. coli aroG-pheA tandem genes into Corynebacterium glutamicum tyrA locus and its effect on L-phenylalanine biosynthesis.

AIM: To study the effect of integration of tandem aroG-pheA genes into the tyrA locus of Corynebacterium glutamicum (C. glutamicum) on the production of L-phenylalanine. METHODS: By nitrosoguanidine mutagenesis, five p-fluorophenylalanine (FP)-resistant mutants of C.glutamicum FP were selected. The tyrA gene encoding prephenate dehydrogenase (PDH) of C.glutamicum was amplified by polymerase chain reaction (PCR) and cloned on the plasmid pPR. Kanamycin resistance gene (Km) and the P(BF) -aroG-pheA-T (GA) fragment of pGA were inserted into tyrA gene to form targeting vectors pTK and pTGAK, respectively. Then, they were transformed into C.glutamicum FP respectively by electroporation. Cultures were screened by a medium containing kanamycin and detected by PCR and phenotype analysis. The transformed strains were used for L-phenylalanine fermentation and enzyme assays. RESULTS: Engineering strains of C.glutamicum (Tyr(-)) were obtained. Compared with the original strain, the transformed strain C. glutamicum GAK was observed to have the highest elevation of L-phenylalanine production by a 1.71-fold, and 2.9-, 3.36-, and 3.0-fold in enzyme activities of chorismate mutase, prephenate dehydratase and 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase, respectively. CONCLUSION: Integration of tandem aroG-pheA genes into tyrA locus of C. glutamicum chromosome can disrupt tyrA gene and increase the yield of L-phenylalanine production.

Expression differentiation of CYC-like floral symmetry genes correlated with their protein sequence divergence in Chirita heterotricha (Gesneriaceae).

CYCLOIDIEA (CYC) and its homologues have been studied intensively in the model organism Antirrhinum majus and related species regarding their function in controlling floral dorsoventral (adaxial-abaxial) asymmetry, including aborting the adaxial and lateral stamens. This raises the question whether the same mechanism underlies the great morphological diversity of zygomorphy in angiosperms, especially in Lamiales sensu lato, a major clade predominantly with zygomorphic flowers. To address this, we selected a representative in Gesneriaceae, the sister to the remainder of Lamiales s.l., to isolate CYC homologues and further investigate their expression patterns using locus-specific semiquantitative reverse transcriptase polymerase chain reaction. Our results showed that four CYC homologues in Chirita heterotricha differentiated spatially and temporally in expression, in which ChCYC1D was only expressed in the adaxial regions, and transcripts of ChCYC1C were distributed in both the adaxial and lateral regions, while ChCYC2A and ChCYC2B transcripts were only detected in the young inflorescences. ChCYC1C expression in the lateral regions correlated with abortion of the lateral stamens in C. heterotricha hinted at its gain of function, i.e., expanding from the adaxial to the lateral regions in expression. Correlatively, the protein sequences of ChCYC genes exhibited remarkable divergences, in which some lineage-specific amino acids between GCYC1 and GCYC2 in conserved functional domains and two sublineage-specific motifs between GCYC1C and GCYC1D in GCYC1 genes had further been identified. Our results indicated that ChCYC genes had probably undergone an expressional differentiation and specialization in establishing the floral dorsoventral asymmetry in C. heterotricha responding to different selective pressure after gene duplication.