ROLE OF P33 IN TOMBUSVIRUS REPLICATION

Replication of the nonsegmented, plus-stranded RNA genome of Cucumber necrosis tombusvirus (CNV) requires two essential overlapping viral-coded replication proteins, the p33 replication co-factor and the p92 RNA-dependent RNA polymerase. In my thesis I describe (i) the effect of phosphorylation of p33, (ii) the RNA chaperone-like activity of p33, and (iii) the role of HSP70s a host proteins in the viral replication. To test the effect of phosphorylation on p33 function, I used in vitro phosphorylated p33. I found that phosphorylation inhibited the ability of p33 to bind to the viral RNA. Phosphorylation-mimicking mutations rendered p33 nonfunctional in plant protoplasts and in yeast. Based on these results, I propose that the primary function of phosphorylation of p33 is to regulate its RNA binding capacity, which could affect the assembly of new viral replicase complexes, recruitment of the viral RNA template into replication and/or release of viral RNA from replication. Thus, phosphorylation of p33 might help in switching the role of the viral RNA from replication to other processes, such as viral RNA encapsidation and cell-to-cell movement. Small plus-stranded RNA viruses do not code for RNA helicases that would facilitate the proper folding of viral RNAs during replication. Instead, small RNA viruses might use RNA chaperones for replication as shown here for the p33 replication protein. In vitro experiments demonstrated that the purified recombinant p33 facilitated RNA synthesis on plusstranded and double-stranded (ds)RNA templates up to 5-fold. In addition, p33 rendered dsRNA templates sensitive to single-strand specific S1 nuclease, suggesting that p33 can destabilize highly structured RNA. Altogether, the RNA chaperone activity of p33 might perform similar biological functions to the helicases. SSa a yeast HSP70 found in the viral replication complex and shown to facilitate viral replication (Serva and Nagy, 2006)To dissect the mode of action of SSA in the viral replication I used temperature sensitive and deletion mutants. Both showed miss localization of p33 compared to the wild type. Purified SSA rendered non functional bacterial expressed p92 functional in an in vitro replication assay. SSa might play a role in the transportation and assembly of viral replication proteins

Plants and Plant Products Useful for Biofuel Manufacture and Feedstock, and Methods of Producing Same

A method of processing plant cellulose includes providing plant cellulose that is from a plant expressing a CESA polypeptide variant having at least one amino acid mutation in its carboxy-terminal transmembrane region; and saccharifying the plant cellulose to produce fermentable sugars. The method can also include fermenting the fermentable sugars to produce alcohol. A method of producing a plant having beneficial saccharification properties includes introducing into a plant a polynucleotide encoding a CESA polypeptide variant having at least one amino acid mutation in its carboxy-terminal transmembrane region; and expressing in the plant the CESA polypeptide variant, wherein plant cellulose of the plant expressing the CESA polypeptide variant has beneficial saccharification properties as compared to a wild-type plant

[\u3csup\u3e14\u3c/sup\u3eC] Glucose Cell Wall Incorporation Assay for the Estimation of Cellulose Biosynthesis

Cellulose is synthesized by Cellulose Synthase A proteins at the plasma membrane using the substrate UDP glucose. Herein, we provide a detailed method for measuring the incorporation of radiolabeled glucose into the cellulose fraction of the cell wall. In this method Arabidopsis seedlings are treated for 2 h with a cellulose biosynthesis inhibitor in the presence of radiolabeled glucose, and are subsequently boiled in acetic-nitric acid to solubilize non-cellulosic material. The radiolabeled glucose detected in the insoluble fraction indicates the amount of cellulose synthesized during the experimental timeframe. The short-term nature of this method is a useful tool in determining if inhibition of cellulose biosynthesis is the herbicides primary mode of action

Identification and thermochemical analysis of high-lignin feedstocks for biofuel and biochemical production

Background - Lignin is a highly abundant biopolymer synthesized by plants as a complex component of plant secondary cell walls. Efforts to utilize lignin-based bioproducts are needed. Results - Herein we identify and characterize the composition and pyrolytic deconstruction characteristics of high-lignin feedstocks. Feedstocks displaying the highest levels of lignin were identified as drupe endocarp biomass arising as agricultural waste from horticultural crops. By performing pyrolysis coupled to gas chromatography-mass spectrometry, we characterized lignin-derived deconstruction products from endocarp biomass and compared these with switchgrass. By comparing individual pyrolytic products, we document higher amounts of acetic acid, 1-hydroxy-2-propanone, acetone and furfural in switchgrass compared to endocarp tissue, which is consistent with high holocellulose relative to lignin. By contrast, greater yields of lignin-based pyrolytic products such as phenol, 2-methoxyphenol, 2-methylphenol, 2-methoxy-4-methylphenol and 4-ethyl-2-methoxyphenol arising from drupe endocarp tissue are documented. Conclusions - Differences in product yield, thermal decomposition rates and molecular species distribution among the feedstocks illustrate the potential of high-lignin endocarp feedstocks to generate valuable chemicals by thermochemical deconstruction

Host transcription factor Rpb11p affects tombusvirus replication and recombination via regulating the accumulation of viral replication proteins

AbstractPrevious genome-wide screens identified over 100 host genes whose deletion/down-regulation affected tombusvirus replication and 32 host genes that affected tombusvirus RNA recombination in yeast, a model host for replication of Tomato bushy stunt virus (TBSV). Down-regulation of several of the identified host genes affected the accumulation levels of p33 and p92pol replication proteins, raising the possibility that these host factors could be involved in the regulation of the amount of viral replication proteins and, thus, they are indirectly involved in TBSV replication and recombination. To test this model, we developed a tightly regulated expression system for recombinant p33 and p92pol replication proteins in yeast. We demonstrate that high accumulation level of p33 facilitated efficient viral RNA replication, while the effect of p33 level on RNA recombination was less pronounced. On the other hand, high level of p92pol accumulation promoted TBSV RNA recombination more efficiently than RNA replication. As predicted, Rpb11p, which is part of the polII complex, affected the accumulation levels of p33 and p92pol as well as altered RNA replication and recombination. An in vitro assay with the tombusvirus replicase further supported that Rpb11p affects TBSV replication and recombination only indirectly, via regulating p33 and p92pol levels. In contrast, the mechanism by which Rpt4p endopeptidase/ATPase and Mps1p threonine/tyrosine kinase affect TBSV recombination is different from that proposed for Rpb11p. We propose a model that the concentration (molecular crowding) of replication proteins within the viral replicase is a factor affecting viral replication and recombination

A Key Role for Heat Shock Protein 70 in the Localization and Insertion of Tombusvirus Replication Proteins to Intracellular Membranes▿

Plus-stranded RNA viruses coopt host proteins to promote their robust replication in infected hosts. Tomato bushy stunt tombusvirus (TBSV) is a model virus that can replicate a small replicon RNA in Saccharomyces cerevisiae and in plants. The tombusvirus replicase complex contains heat shock protein 70 (Hsp70), an abundant cytosolic chaperone, which is required for TBSV replication. To dissect the function of Hsp70 in TBSV replication, in this paper we use an Hsp70 mutant (ssa1 ssa2) yeast strain that supports a low level of TBSV replication. Using confocal laser microscopy and cellular fractionation experiments, we find that the localization of the viral replication proteins changes to the cytosol in the mutant cells from the peroxisomal membranes in wild-type cells. An in vitro membrane insertion assay shows that Hsp70 promotes the integration of the viral replication proteins into subcellular membranes. This step seems to be critical for the assembly of the viral replicase complex. Using a gene-silencing approach and quercetin as a chemical inhibitor to downregulate Hsp70 levels, we also confirm the significance of cytosolic Hsp70 in the replication of TBSV and other plant viruses in a plant host. Taken together, our results suggest that cytosolic Hsp70 plays multiple roles in TBSV replication, such as affecting the subcellular localization and membrane insertion of the viral replication proteins as well as the assembly of the viral replicase

Subfunctionalization of Cellulose Synthases in Seed Coat Epidermal Cells Mediates Secondary Radial Wall Synthesis and Mucilage Attachment

Arabidopsis (Arabidopsis thaliana) epidermal seed coat cells follow a complex developmental program where, following fertilization, cells of the ovule outer integument differentiate into a unique cell type. Two hallmarks of these cells are the production of a doughnut-shaped apoplastic pocket filled with pectinaceous mucilage and the columella, a thick secondary cell wall. Cellulose is thought to be a key component of both these secondary cell wall processes. Here, we investigated the role of cellulose synthase (CESA) subunits CESA2, CESA5, and CESA9 in the seed coat epidermis. We characterized the roles of these CESA proteins in the seed coat by analyzing cell wall composition and morphology in cesa mutant lines. Mutations in any one of these three genes resulted in lower cellulose content, a loss of cell shape uniformity, and reduced radial wall integrity. In addition, we found that attachment of the mucilage halo to the parent seed following extrusion is maintained by cellulose-based connections requiring CESA5. Hence, we show that cellulose fulfills an adhesion role between the extracellular mucilage matrix and the parent cell in seed coat epidermal cells. We propose that mucilage remains attached to the seed coat through interactions between components in the seed mucilage and cellulose. Our data suggest that CESA2 and CESA9 serve in radial wall reinforcement, as does CESA5, but CESA5 also functions in mucilage biosynthesis. These data suggest unique roles for different CESA subunits in one cell type and illustrate a complex role for cellulose biosynthesis in plant developmental biology