The WD40-Repeat Proteins NFC101 and NFC102 Regulate Different Aspects of Maize Development through Chromatin Modification

The maize (Zea mays) nucleosome remodeling factor complex component101 (nfc101) and nfc102 are putative paralogs encoding WD-repeat proteins with homology to plant and mammalian components of various chromatin modifying complexes. In this study, we generated transgenic lines with simultaneous nfc101 and nfc102 downregulation and analyzed phenotypic alterations, along with effects on RNA levels, the binding of NFC101/NFC102, and Rpd3-type histone deacetylases (HDACs), and histone modifications at selected targets. Direct NFC101/NFC102 binding and negative correlation with mRNA levels were observed for indeterminate1 (id1) and the florigen Zea mays CENTRORADIALIS8 (ZCN8), key activators of the floral transition. In addition, the abolition of NFC101/NFC102 association with repetitive sequences of different transposable elements (TEs) resulted in tissue-specific upregulation of nonpolyadenylated RNAs produced by these regions. All direct nfc101/nfc102 targets showed histone modification patterns linked to active chromatin in nfc101/nfc102 downregulation lines. However, different mechanisms may be involved because NFC101/NFC102 proteins mediate HDAC recruitment at id1 and TE repeats but not at ZCN8. These results, along with the pleiotropic effects observed in nfc101/nfc102 downregulation lines, suggest that NFC101 and NFC102 are components of distinct chromatin modifying complexes, which operate in different pathways and influence diverse aspects of maize development.     

Expression of Lex antigen in Schistosoma japonicum and S.haematobium and immune responses to Lex in infected animals: lack of Lex expression in other trematodes and nematodes

Adults of the human parasitic trematode Schistosoma mansoni, which causes hepatosplenic/intestinal complications in humans, synthesize glycoconjugates containing the Lewis x (Lex) Galbeta1-->4(Fucalpha1-- >3)GlcNAcbeta1-->R, but not sialyl Lewis x (sLex), antigen. We now report on our analyses of Lexand sLexexpression in S.haematobium and S.japonicum, which are two other major species of human schistosomes that cause disease, and the possible autoimmunity to these antigens in infected individuals. Antigen expression was evaluated by both ELISA and Western blot analyses of detergent extracts of parasites using monoclonal antibodies. Several high molecular weight glycoproteins in both S. haematobium and S. japonicum contain the Lexantigen, but no sialyl Lexantigen was detected. In addition, sera from humans and rodents infected with S.haematobium and S.japonicum contain antibodies reactive with Lex. These results led us to investigate whether Lexantigens are expressed in other helminths, including the parasitic trematode Fasciola hepatica , the parasitic nematode Dirofilaria immitis (dog heartworm), the ruminant nematode Haemonchus contortus , and the free-living nematode Caenorhabditis elegans . Neither Lexnor sialyl-Lexis detectable in these other helminths. Furthermore, none of the helminths, including schistosomes, express Lea, Leb, Ley, or the H- type 1 antigen. However, several glycoproteins from all helminths analyzed are bound by Lotus tetragonolobus agglutinin , which binds Fucalpha1-->3GlcNAc, and Wisteria floribunda agglutinin, which binds GalNAcbeta1-->4GlcNAc (lacdiNAc or LDN). Thus, schistosomes may be unique among helminths in expressing the Lexantigen, whereas many different helminths may express alpha1,3-fucosylated glycans and the LDN motif.      

Differential expression of chitin synthase III and IV mRNAs in ascomata of Tuber borchii Vittad

A full-length genomic clone encoding a class III chitin synthase (CHS) and one DNA fragment corresponding to a class IV CHS were isolated from the mycorrhizal fungus Tuber borchii and used for an extensive expression analysis, together with a previously identified DNA fragment corresponding to a class II CHS. All three Chs mRNAs are constitutively expressed in vegetative mycelia, regardless of the age, mode of growth, and proliferation capacity of the hyphae. A strikingly different situation was observed in ascomata, where class III and IV, but not class II, mRNAs are differentially expressed in a maturation stage-dependent manner and accumulate, respectively, in sporogenic and vegetative hyphae. These data, the first on the expression of distinct Chs mRNAs during fruitbody development, point to the different cellular roles that can be played by distinct chitin synthases in the differentiation of spores of sexual origin (CHS III) or in ascoma enlargement promoted by the growth of vegetative hyphae (CHS IV).     

Differential Expression of Chitin Synthase III and IV mRNAs in Ascomata of Tuber borchii Vittad

A full-length genomic clone encoding a class III chitin synthase (CHS) and one DNA fragment corresponding to a class IV CHS were isolated from the mycorrhizal fungus Tuber borchii and used for an extensive expression analysis, together with a previously identified DNA fragment corresponding to a class II CHS. All three Chs mRNAs are constitutively expressed in vegetative mycelia, regardless of the age, mode of growth, and proliferation capacity of the hyphae. A strikingly different situation was observed in ascomata, where class III and IV, but not class II, mRNAs are differentially expressed in a maturation stage-dependent manner and accumulate, respectively, in sporogenic and vegetative hyphae. These data, the first on the expression of distinct Chs mRNAs during fruitbody development, point to the different cellular roles that can be played by distinct chitin synthases in the differentiation of spores of sexual origin (CHS III) or in ascoma enlargement promoted by the growth of vegetative hyphae (CHS IV).     

Identification of genes required for de novo DNA methylation in Arabidopsis

De novo DNA methylation in Arabidopsis thaliana is catalyzed by the methyltransferase DRM2, a homolog of the mammalian de novo methyltransferase DNMT3. DRM2 is targeted to DNA by small interfering RNAs (siRNAs) in a process known as RNA-directed DNA Methylation (RdDM). While several components of the RdDM pathway are known, a functional understanding of the underlying mechanism is far from complete. We employed both forward and reverse genetic approaches to identify factors involved in de novo methylation. We utilized the FWA transgene, which is methylated and silenced when transformed into wild-type plants, but unmethylated and expressed when transformed into de novo methylation mutants. Expression of FWA is marked by a late-flowering phenotype, which is easily scored in mutant versus wild-type plants. By reverse genetics we discovered the requirement for known RdDM effectors AGO6 and NRPE5a for efficient de novo methylation. A forward genetic approach uncovered alleles of several components of the RdDM pathway, including alleles of clsy1, ktf1 and nrpd/e2, which have not been previously shown to be required for the initial establishment of DNA methylation. Mutations were mapped and genes cloned by both traditional and whole genome sequencing approaches. The methodologies and the mutant alleles discovered will be instrumental in further studies of de novo DNA methylation.Key words: DNA methylation, Arabidopsis, de novo, genetic screen, whole-genome sequencing     

The human immunodeficiency virus antigen Nef forms protein bodies in leaves of transgenic tobacco when fused to zeolin

Protein bodies (PB) are stable polymers naturally formed by certain seed storage proteins within the endoplasmic reticulum (ER). The human immunodeficiency virus negative factor (Nef) protein, a potential antigen for the development of an anti-viral vaccine, is highly unstable when introduced into the plant secretory pathway, probably because of folding defects in the ER environment. The aim of this study was to promote the formation of Nef-containing PB in tobacco (Nicotiana tabacum) leaves by fusing the Nef sequence to the N-terminal domains of the maize storage protein gamma-zein or to the chimeric protein zeolin (which efficiently forms PB and is composed of the vacuolar storage protein phaseolin fused to the N-terminal domains of gamma-zein). Protein blots and pulse-chase indicate that fusions between Nef and the same gamma-zein domains present in zeolin are degraded by ER quality control. Consistently, a mutated zeolin, in which wild-type phaseolin was substituted with a defective version known to be degraded by ER quality control, is unstable in plant cells. Fusion of Nef to the entire zeolin sequence instead allows the formation of PB detectable by electron microscopy and subcellular fractionation, leading to zeolin-Nef accumulation higher than 1% of total soluble protein, consistently reproduced in independent transgenic plants. It is concluded that zeolin, but not its gamma-zein portion, has a positive dominant effect over ER quality control degradation. These results provide insights into the requirements for PB formation and avoidance of quality-control degradation, and indicate a strategy for enhancing foreign protein accumulation in plants.