Arabidopsis RecQI4A suppresses homologous recombination and modulates DNA damage responses.

The DNA damage response and DNA recombination are two interrelated mechanisms involved in maintaining the integrity of the genome, but in plants they are poorly understood. RecQ is a family of genes with conserved roles in the regulation of DNA recombination in eukaryotes; there are seven members in Arabidopsis. Here we report on the functional analysis of the Arabidopsis RecQl4A gene. Ectopic expression of Arabidopsis RecQl4A in yeast RecQ-deficient cells suppressed their hypersensitivity to the DNA-damaging drug methyl methanesulfonate (MMS) and enhanced their rate of homologous recombination (HR). Analysis of three recQl4A mutant alleles revealed no obvious developmental defects or telomere deregulation in plants grown under standard growth conditions. Compared with wild-type Arabidopsis, the recQl4A mutant seedlings were found to be hypersensitive to UV light and MMS, and more resistant to mitomycin C. The average frequency of intrachromosomal HR in recQl4A mutant plants was increased 7.5-fold over that observed in wild-type plants. The data reveal roles for Arabidopsis RecQl4A in maintenance of genome stability by modulation of the DNA damage response and suppression of HR.

Arabidopsis RecQsim, a plant-specific member of the RecQ helicase family, can suppress the MMS hypersensitivity of the yeast sgs1 mutant.

The Arabidopsis genome contains seven genes that belong to the RecQ family of ATP-dependent DNA helicases. RecQ members in Saccharomyces cerevisiae (SGS1) and man (WRN, BLM and RecQL4) are involved in DNA recombination, repair and genome stability maintenance, but little is known about the function of their plant counterparts. The Arabidopsis thaliana RecQsim gene is remarkably different from the other RecQ-like genes due to an insertion in its helicase domain. We isolated the AtRecQsim orthologues from rice and rape and established the presence of a similar insertion in their helicase domain, which suggests a plant specific function for the insert. The expression pattern of the AtRecQsim gene was compared with the other Arabidopsis RecQ-like members in different tissues and in response to stress. The transcripts of the AtRecQsim gene were found in all plant organs and its accumulation was higher in roots and seedlings, as compared to the other AtRecQ-like members. In contrast to most AtRecQ-like genes, the examined environmental cues did not have a detectable effect on the accumulation of the AtRecQsim transcripts. The budding yeast sgs1 mutant, which is known to be hypersensitive to the DNA-damaging drug MMS, was transformed with the AtRecQsim cDNA. The AtRecQsim gene suppressed the MMS hypersensitivity phenotype of the sgs1 cells. We propose that the Arabidopsis RecQsim gene, despite its unusual structure, exhibits an evolutionary conserved function.

The major Cu,Zn SOD of the phytopathogen Claviceps purpurea is not essential for pathogenicity.

summary Superoxide dismutase (SOD) activities of the biotrophic pathogen Claviceps purpurea, which causes the ergot disease on a wide range of host grasses, were examined in axenic and pathogenic cultures. Almost all SOD activity in axenic culture originated from a single Cu,Zn SOD; a substantial part of this activity could be separated from lyophilized intact mycelia by gentle washing, indicating that this protein is at least partially secreted. The corresponding gene (cpsod1) was cloned and characterized; like other fungal Cu,Zn SOD genes, it groups with the extracellular mammalian Cu,Zn SODs in a phylogenetic tree. Northern analyses showed that cpsod1 is strongly induced by copper and weakly induced by iron; superoxide generated by paraquat, or xanthine and xanthine oxidase, as well as hydrogen peroxide, had no effect on gene expression under axenic conditions. Analysis of the deletion mutant Deltacpsod1 showed that, although growth in axenic culture was generally slower, sensitivity to paraquat was not increased in comparison to the wild-type. Pathogenicity assays showed that this gene is not essential for parasitic growth in rye; no further soluble SOD activity is induced in the mutant.

Identification of three differentially expressed hydrophobins in Pleurotus ostreatus (oyster mushroom).

Three proteins with characteristic features of class I hydrophobins, designated POH1, POH2 and POH3, were isolated from the basidiomycete Pleurotus ostreatus. Based on N-terminal sequence analyses, their cDNAs were isolated using RT-PCR; the cDNAs and corresponding genes were sequenced and their regulation studied. POH1 is expressed in the fruiting bodies but not in vegetative mycelium. The regulation of POH2 and POH3 is tightly correlated. Both genes are switched off in the fruiting bodies but abundantly expressed in the vegetative mycelium of both monokaryon and dikaryon. POH2 and POH3 were isolated from the culture medium and from aerial hyphae. Co-purified POH2 and POH3 assembled in vitro into a protein membrane with a typical rodlet pattern as found previously with other hydrophobins. Similar structures were detected on the surface of aerial hyphae.

An abundant hydrophobin (ABH1) forms hydrophobic rodlet layers in Agaricus bisporus fruiting bodies.

The SDS-insoluble protein fraction of Agaricus bisporus fruiting bodies was solubilized with trifluoroacetic acid. On SDS-PAGE this fraction was found to contain one abundant protein with an apparent M(r) of 16 kDa. The N-terminal amino acid sequence of this protein was determined and RT-PCR used to isolate a cDNA clone which upon sequencing identified the protein as a typical class I hydrophobin (ABH1). The gene (ABH1) was isolated and sequenced, and a second hydrophobin gene (ABH2) was found about 2.5 kbp downstream of ABH1. Purified ABH1 self-assembled at hydrophobic-hydrophilic interfaces, producing the typical rodlet layer known from other hydrophobins. Similar rodlets were observed on the surface of the fruiting body, while immunological localization showed the hydrophobin to be particularly abundant at the outer surface of fruiting bodies, in the veil and in the core tissue of the stipe. Transcripts of ABH1 were found only in fruiting-body hyphae. The ABH1 hydrophobin is probably solely responsible for the hydrophobicity of the fruiting-body surface but may also line air channels within fruiting bodies.