Induction of a Citrus gene highly homologous to plant and yeast thi genes involved in thiamine biosynthesis during natural and ethylene-induced fruit maturation

Maturing citrus fruit undergo pigment changes which can be enhanced by exogenous ethylene. In order to identify genes induced by ethylene in citrus fruit peel, we cloned the gene c-thi1. mRNA corresponding to c-thi1 increased gradually in the peel during natural fruit maturation and in response to ethylene. GA₃ pretreatment reduced the inductive effect of ethylene. Levels of c-thi1 increased also in juice sacs but the effect of ethylene was much less prominent. c-thi1 is homologous to yeast and plant genes encoding for an enzyme belonging to the pathway of thiamine biosynthesis. The data suggest that thiamine is involved in citrus fruit maturation.     

Differential expression of the putative Kex2 processed and secreted aspartic proteinase gene family of Cryphonectria parasitica

Kex2-silenced strains of Cryphonectria parasitica, the ascomycete causal agent of chestnut blight, show a significant reduction in virulence, reduced sexual and asexual sporulation and reductions in mating and fertility. Due to this and the known involvement of Kex2 in the processing of important proproteins in other systems, we searched the whole C. parasitica genome for putative Kex2 substrates. Out of 1299 open reading frames (ORFs) predicted to be secreted, 222 ORFs were identified as potential Kex2 substrates by this screen. Within the putative substrates we identified cell wall modifying proteins, putative proteinases, lipases, esterases, and oxidoreductases. This in silico screen also uncovered a family of nine secreted aspartic proteinases (SAPs) of C. parasitica. Northern blot analyses of this gene family showed differential expression when exposed to chestnut wood and Cryphonectria hypovirus 1 (CHV1). Due to the reduction in fungal virulence known to be caused upon hypoviral infection of C. parasitica, the differential gene expression observed, and the known involvement of SAPs in virulence in other systems, we conducted deletion analyses of four of these proteinases, representing different expression patterns. Deletion of each of the four SAPs did not affect growth rates, sporulation or virulence, suggesting that none of the considered SAPs is essential for the full development or virulence of C. parasitica under the conditions tested.     

Cellulose biosynthesis in plants: from genes to rosettes

Modern techniques of gene cloning have identified the CesA genes as encoding the probable catalytic subunits of the plant CelS, the cellulose synthase enzyme complex visualized in the plasma membrane as rosettes. At least 10 CesA isoforms exist in Arabidopsis and have been shown by mutant analyses to play distinct role/s in the cellulose synthesis process. Functional specialization within this family includes differences in gene expression, regulation and, possibly, catalytic function. Current data points towards some CesA isoforms potentially being responsible for initiation or elongation of the recently identified sterol beta-glucoside primer within different cell types, e.g. those undergoing either primary or secondary wall cellulose synthesis. Different CesA isoforms may also play distinct roles within the rosette, and there is some circumstantial evidence that CesA genes may encode the catalytic subunit of the mixed linkage glucan synthase or callose synthase. Various other proteins such as the Korrigan endocellulase, sucrose synthase, cytoskeletal components, Rac13, redox proteins and a lipid transfer protein have been implicated to be involved in synthesizing cellulose but, apart from CesAs, only Korrigan has been definitively linked with cellulose synthesis. These proteins should prove valuable in identifying additional CelS components.     

Mycovirus cryphonectria hypovirus 1 elements cofractionate with trans-Golgi network membranes of the fungal host Cryphonectria parasitica

The mycovirus cryphonectria hypovirus 1 (CHV1) causes proliferation of vesicles in its host, Cryphonectria parasitica, the causal agent of chestnut blight. These vesicles have previously been shown to contain both CHV1 genomic double-stranded RNA (dsRNA) and RNA polymerase activity. To determine the cellular origins of these virus-induced membrane structures, we compared the fractionation of several cellular and viral markers. Results showed that viral dsRNA, helicase, polymerase, and protease p29 copurify with C. parasitica trans-Golgi network (TGN) markers, suggesting that the virus utilizes the fungal TGN for replication. We also show that the CHV1 protease p29 associates with vesicle membranes and is resistant to treatments that would release peripheral membrane proteins. Thus, p29 behaves as an integral membrane protein of the vesicular fraction derived from the fungal TGN. Protease p29 was also found to be fully susceptible to proteolytic digestion in the absence of detergent and, thus, is wholly or predominantly on the cytoplasmic face of the vesicles. Fractionation analysis of p29 deletion variants showed that sequences in the C terminal of p29 mediate membrane association. In particular, the C-terminal portion of the protein (Met-135-Gly-248) is sufficient for membrane association and is enough to direct p29 to the TGN vesicles in the absence of other viral elements.