Characterisation of epitopes on barley mild mosaic virus coat protein recognised by a panel of novel monoclonal antibodies.

Barley mild mosaic virus (BaMMV), a member of the family Potyviridae, genus Bymovirus, is involved in the economically important yellow mosaic disease of winter barley in East Asia and Europe. We investigated serological properties of bacterially expressed BaMMV coat protein (CP) of a German isolate. Ten mouse monoclonal antibodies were produced using purified E. coli expressed BaMMV-CP as immunogen. The reactivity of MAbs with different strains of BaMMV was analysed by several immunological methods that are frequently used in diagnostic virology: enzyme-linked immunosorbent assay (ELISA), dot-blot, Western-blotting (WB), direct tissue blotting immunoassay (DTBIA) and immunoelectron microscopy (IEM). The amino acids involved in the formation of epitopes recognised by several MAbs were mapped by using synthetic pin-bound peptides and the localisation of epitopes in assembled virus particles was determined by electron microscope studies. MAbs V29 and M1 decorated the whole virion indicating that their epitopes 6PDPI9 and 96ITDDEK101, respectively, are exposed on the surface. The MAbs V6 and V14 both interacted with 44LPEPKM49, which seems to be accessible at only one end of the virus particle. The MAbs V6, V14, V29 and M1 detected epitopes common to a wide range of BaMMV isolates and can therefore be used effectively in routine diagnostic tests for BaMMV from barley leaves. We suggest that MAbs M1, V6, V14 and V29 are most suitable for use in TAS-ELISA, V6, V14 and V29 for Western blotting and V29 and M1 for electron microscope serology.

Divergence of eukaryotic secretory components: the Candida albicans homolog of the Saccharomyces cerevisiae ++Sec20 protein is N terminally truncated, and its levels determine antifungal drug resistance and growth.

Sec20p is a component of the yeast Saccharomyces cerevisiae secretory pathway that does not have a close homolog in higher eukaryotic cells. To verify the function of Sec20p in other fungal species, we characterized the gene encoding a Sec20p homolog in the human fungal pathogen Candida albicans. The deduced protein has 27% identity with, but is missing about 100 N-terminal residues compared to S. cerevisiae Sec20p, which is part of the cytoplasmic tail interacting with the cytoplasmic protein Tip20p. Because a strain lacking both C. albicans SEC20 alleles could not be constructed, we placed SEC20 under transcriptional control of two regulatable promoters, MET3p and PCK1p. Repression of SEC20 expression in these strains prevented (MET3p-SEC20 allele) or retarded (PCK1p-SEC20 allele) growth and led to the appearance of extensive intracellular membranes, which frequently formed stacks. Reduced SEC20 expression in the PCK1p-SEC20 strain did not affect morphogenesis but led to a series of hypersensitivity phenotypes including supersensitivity to aminoglycoside antibiotics, to nystatin, to sodium dodecyl sulfate, and to cell wall inhibitors. These results demonstrate the occurrence and function of Sec20p in a fungal species other than S. cerevisiae, but the lack of the N-terminal domain and the apparent absence of a close TIP20 homolog in the C. albicans genome also indicate a considerable diversity in mechanisms of retrograde vesicle traffic in eukaryotes.

Differential accumulation of the 10-, 16- and 23-kDa peripheral components of the water-splitting complex of photosystem II in mesophyll and bundle-sheath chloroplasts of the dicotyledonous C4 plant Flaveria trinervia (Spreng.) C. Mohr.

The differential expression of PSII genes was investigated in mesophyll and bundle-sheath cells of Flaveria trinervia, a dicotyledonous C4 plant of the NADP-malic enzyme type. A comprehensive immunoblot analysis showed that three extrinsic proteins of the water-splitting complex (10, 16 and 23 kDa) are selectively depleted in mature bundle-sheath chloroplasts. In contrast, the reaction-centre core remained virtually unaffected as inferred from the abundance of the 47-kDa chlorophyll-a-binding protein, the D1 and D2 polypeptides, cytochrome b559 and the 34-kDa polypeptide. The selective depletion of the 10-, 16- and 23-kDa polypeptides in bundle-sheath chloroplasts was paralleled by a diminished PSII capacity. On the basis of oxygen evolution in the presence of the artifical electron acceptor 2,5-dimethyl-p-benzoquinone, bundle-sheath chloroplasts maintained up to 23 % of the PSII capacity shown by mesophyll chloroplasts. However, the levels of the 10-, 16- and 23-kDa proteins and, concomitantly, PSII activity varied to some degree and appeared to be correlated with environmental factors caused by seasonal changes. The selective depletion of the three members of the water-splitting complex was not reflected at the transcript level. The corresponding mRNAs were detectable in considerable amounts in bundle-sheath cells, indicating that the depletion of these proteins is regulated by post-transcriptional events. These findings reinforce the view that the peripheral proteins of the water-splitting complex are a focal point for controlling PSII activity in bundle-sheath chloroplasts of both mono- and dicotyledonous C4 plants of the NADP-malic enzyme subtype.

The mitochondrial complex in Cryptophyceae.

The unitary nature of the mitochondrion and the characteristic flattened finger-like morphology of the cristae were demonstrated in the Cryptophyceae. Hemiselmis rufescens contained an unbranched vermiform mitochondrion in contrast to the variously branched complex. comprising an interconnected peripheral and central reticulum, in Chroomonas sp. and strains of Cryptomonas. The systematic value of the shape and distribution of the mitochondria in the examined genera was suggested.