Jeffrey P. Woessner

Gene discovery and gene function assignment in filamentous fungi

Filamentous fungi are a large group of diverse and economically important microorganisms. Large-scale gene disruption strategies developed in budding yeast are not applicable to these organisms because of their larger genomes and lower rate of targeted integration (TI) during transformation. We developed transposon-arrayed gene knockouts (TAGKO) to discover genes and simultaneously create gene disruption cassettes for subsequent transformation and mutant analysis. Transposons carrying a bacterial and fungal drug resistance marker are used to mutagenize individual cosmids or entire libraries in vitro. Cosmids are annotated by DNA sequence analysis at the transposon insertion sites, and cosmid inserts are liberated to direct insertional mutagenesis events in the genome. Based on saturation analysis of a cosmid insert and insertions in a fungal cosmid library, we show that TAGKO can be used to rapidly identify and mutate genes. We further show that insertions can create alterations in gene expression, and we have used this approach to investigate an amino acid oxidation pathway in two important fungal phytopathogens.

Molecular characterization of a zygote wall protein: an extensin-like molecule in Chlamydomonas reinhardtii.

The green alga Chlamydomonas reinhardtii elaborates two biochemically and morphologically distinct cell walls during its life cycle: one surrounds the vegetative and gametic cell and the other encompasses the zygote. Hydroxyproline-rich glycoproteins (HRGPs) constitute a major component of both walls. We describe the isolation and characterization of a zygote-specific gene encoding a wall HRGP. The derived amino acid sequence of this algal HRGP is similar to those of higher plant extensins, rich in proline and serine residues and possessing repeating amino acid motifs, notably X(Pro)3 and (Ser-Pro)n. Antiserum against this zygote wall protein detected common epitopes in several other zygote polypeptides, at least one of which is also encoded by a zygote-specific gene. We conclude that there is one set of HRGP wall genes expressed only in zygotes and another set that is specific to vegetative and gametic cells.

Domain conservation in several volvocalean cell wall proteins

Based on our previous work demonstrating that (SerPro)x epitopes are common to extensin-like cell wall proteins in Chlamydomonas reinhardtii, we looked for similar proteins in the distantly related species C. eugametos. Using a polyclonal antiserum against a (SerPro)10 oligopeptide, we found distinct sets of stage-specific polypeptides immunoprecipitated from in vitro translations of C. eugametos RNA. Screening of a C. eugametos cDNA expression library with the antiserum led to the isolation of a cDNA (WP6) encoding a (SerPro)x-rich multidomain wall protein. Analysis of a similarly selected cDNA (VSP-3) from a C. reinhardtii cDNA expression library revealed that it also coded for a (SerPro)x-rich multidomain wall protein. The C-terminal rod domains of VSP-3 and WP6 are highly homologous, while the N-terminal domains are dissimilar; however, the N-terminal domain of VSP-3 is homologous to the globular domain of a cell wall protein from Volvox carteri. Exon shuffling might be responsible for this example of domain conservation over 350 million years of volvocalean cell wall protein evolution.

Zygote and vegetative cell wall proteins in Chlamydomonas reinhardtii share a common epitope, (SerPro)x

Abstract Polyclonal antibodies directed against a (SerPro) 10 oligopeptide and a s-galactosidase/zygote-wall-gene fusion protein were used in in vitro translation/immunoprecipitation experiments to show that (SerPro) x is a common motif in proteins which assemble into either the zygotic or the vegetative/gametic cell walls of Chlamydomonas reinhardtii . There are at least five, and possibly as many as ten different zygote-specific proteins displaying (SerPro) x -like epitopes. Zygote-specific cDNAs corresponding to two of these proteins have been previously characterized and shown to encode several regions of (SerPro) ranging from 2 to 9 repeats. In addition, the (SerPro) 10 antibody recognizes five vegetative/gamete-specific proteins from cells induced for wall synthesis. One of these five corresponds to the vegetative/gamete-wall glycoprotein GP2, as confirmed by immunoprecipitation with a polyclonal antibody to purified GP2. The GP2 antibody detects epitopes other than (SerPro) x and also immunoprecipitates a novel zygote-specific protein.

A ZYGOTE-SPECIFIC PROTEIN WITH HYDROXYPROLINE-RICH GLYCOPROTEIN DOMAINS AND LECTIN-LIKE DOMAINS INVOLVED IN THE ASSEMBLY OF THE CELL WALL OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)

The cell wall of Chlamydomonas reinhardtii zygotes, which forms rapidly after the fusion of wall‐free gametes, provides a tractable system for studying the properties and assembly of hydroxyproline‐rich glycoproteins, the major proteinaceous components of green algal and plant cell walls. We report the cloning of the zsp2 gene and the analysis of its ZSP‐2 product, a 58.9 kDa polypeptide that is synthesized exclusively by zygotes. The protein contains two (SP)x repeats, establishing it as a member of the cell wall hydroxyproline‐rich glycoproteins family. It also contains a 4‐fold iteration of an amino acid sequence centered around cysteine residues, a configuration found in both plant and animal lectins. Furthermore, we report four observations on pellicle composition and production. First, cell‐free preparations of the pellicle matrix are rich in hydroxyproline, arabinose, and galactose and contain bundles of very long fibrils. Second, glutathione blocks pellicle formation and results in the accumulation of long fibrils in the growth medium. Third, antibody to ZSP‐2 also blocks pellicle formation. Fourth, ZSP‐2 immunolocalizes to the boundary between the outer layers of the wall proper and the pellicle matrix. These observations are consistent with the possibility that the Cys‐rich (glutathione‐sensitive) lectin‐like domains of ZSP‐2 may bind to sugar residues on the long fibrils and anchor them to the cell wall, thereby initiating and maintaining pellicle formation.