The plant formin AtFH4 interacts with both actin and microtubules, and contains a newly identified microtubule-binding domain.

The dynamic behaviour of the actin cytoskeleton in plants relies on the coordinated action of several classes of actin-binding proteins (ABPs). These ABPs include the plant-specific subfamilies of actin-nucleating formin proteins. The model plant species Arabidopsis thaliana has over 20 formin proteins, all of which contain plant-specific regions in place of the GTPase-binding domain, formin homology (FH)3 domain, and DAD and DID motifs found in many fungal and animal formins. We have identified for the first time a plant-specific region of the membrane-integrated formin AtFH4 that mediates an association with the microtubule cytoskeleton. In vitro analysis shows that this region (named the GOE domain) binds directly to microtubules. Overexpressed AtFH4 accumulates at the endoplasmic reticulum membrane and co-aligns the endoplasmic reticulum with microtubules. The FH1 and FH2 domains of formins are conserved in plants, and we show that these domains of AtFH4 nucleate F-actin. Together, these data suggest that the combination of plant-specific and conserved domains enables AtFH4 to function as an interface between membranes and both major cytoskeletal networks.

Use of a pooled transposon mutation grid to demonstrate roles in disease development for Erwinia carotovora subsp. atroseptica putative type III secreted effector (DspE/A) and helper (HrpN) proteins.

Soft rot Erwinia spp., like other closely related plant pathogens, possess a type III secretion system (TTSS) (encoded by the hrp gene cluster) implicated in disease development. We report the sequence of the entire hrp gene cluster and adjacent dsp genes in Erwinia carotovora subsp. atroseptica SCRI1039. The cluster is similar in content and structural organization to that in E. amylovora. However, eight putative genes of unknown function located within the E. carotovora subsp. atroseptica cluster do not have homologues in the E. amylovora cluster. An arrayed set of Tn5 insertional mutants (mutation grid) was constructed and pooled to allow rapid isolation of mutants for any given gene by polymerase chain reaction screening. This novel approach was used to obtain mutations in two structural genes (hrcC and hrcV), the effector gene dspE/A, and the helper gene hrpN. An improved pathogenicity assay revealed that these mutations led to significantly reduced virulence, showing that both the putative E. carotovora subsp. atroseptica TTSS-delivered effector and helper proteins are required for potato infection.

Soft rot erwiniae: from genes to genomes.

UNLABELLED: SUMMARY The soft rot erwiniae, Erwinia carotovora ssp. atroseptica (Eca), E. carotovora ssp. carotovora (Ecc) and E. chrysanthemi (Ech) are major bacterial pathogens of potato and other crops world-wide. We currently understand much about how these bacteria attack plants and protect themselves against plant defences. However, the processes underlying the establishment of infection, differences in host range and their ability to survive when not causing disease, largely remain a mystery. This review will focus on our current knowledge of pathogenesis in these organisms and discuss how modern genomic approaches, including complete genome sequencing of Eca and Ech, may open the door to a new understanding of the potential subtlety and complexity of soft rot erwiniae and their interactions with plants. TAXONOMY: The soft rot erwiniae are members of the Enterobacteriaceae, along with other plant pathogens such as Erwinia amylovora and human pathogens such as Escherichia coli, Salmonella spp. and Yersinia spp. Although the genus name Erwinia is most often used to describe the group, an alternative genus name Pectobacterium was recently proposed for the soft rot species. HOST RANGE: Ech mainly affects crops and other plants in tropical and subtropical regions and has a wide host range that includes potato and the important model host African violet (Saintpaulia ionantha). Ecc affects crops and other plants in subtropical and temperate regions and has probably the widest host range, which also includes potato. Eca, on the other hand, has a host range limited almost exclusively to potato in temperate regions only. Disease symptoms: Soft rot erwiniae cause general tissue maceration, termed soft rot disease, through the production of plant cell wall degrading enzymes. Environmental factors such as temperature, low oxygen concentration and free water play an essential role in disease development. On potato, and possibly other plants, disease symptoms may differ, e.g. blackleg disease is associated more with Eca and Ech than with Ecc. USEFUL WEBSITES: http://www.scri.sari.ac.uk/TiPP/Erwinia.htm, http://www.ahabs.wisc.edu:16080/ approximately pernalab/erwinia/index.htm, http://www.tigr.org/tdb/mdb/mdbinprogress.html, http://www.sanger.ac.uk/Projects/E_carotovora/.

Sample sequencing of a selected region of the genome of Erwinia carotovora subsp. atroseptica reveals candidate phytopathogenicity genes and allows comparison with Escherichia coli.

Genome sequencing is making a profound impact on microbiology. Currently, however, only one plant pathogen genome sequence is publicly available and no genome-sequencing project has been initiated for any species of the genus Erwinia, which includes several important plant pathogens. This paper describes a targeted sample sequencing approach to study the genome of Erwinia carotovora subsp. atroseptica (Eca), a major soft-rot pathogen of potato. A large insert DNA (approx. 115 kb) library of Eca was constructed using a bacterial artificial chromosome (BAC) vector. Hybridization and end-sequence data revealed two overlapping BAC clones that span an entire hrp gene cluster. Random subcloning and one-fold sequence coverage (>200 kb) across these BACs identified 25 (89%) of 28 hrp genes predicted from the orthologous hrp cluster of Erwinia amylovora. Regions flanking the hrp cluster contained orthologues of known or putative pathogenicity operons from other Erwinia species, including dspEF (E. amylovora), hecAB and pecSM (E. chrysanthemi), sequences similar to genes from the plant pathogen Xylella fastidiosa, including haemagglutinin-like genes, and sequences similar to genes involved in rhizobacterium-plant interactions. Approximately 10% of the sequences showed strongest nucleotide similarities to genes in the closely related model bacterium and animal pathogen Escherichia coli. However, the positions of some of these genes were different in the two genomes. Approximately 30% of sequences showed no significant similarity to any database entries. A physical map was made across the genomic region spanning the hrp cluster by hybridization to the BAC library and to digested BAC clones, and by PCR between sequence contigs. A multiple genome coverage BAC library and one-fold sample sequencing are an effective combination for extracting useful information from important regions of the Eca genome, providing a wealth of candidate novel pathogenicity genes for functional analyses. Other genomic regions could be similarly targeted.