The Aspergillus nidulans IQGAP orthologue SepG is required for constriction of the contractile actomyosin ring.

In this research we report that the sepG1 mutation in Aspergillus nidulans resides in gene AN9463, which is predicted to encode an IQGAP orthologue. The genetic lesion is predicted to result in a G-to-R substitution at residue 1637 of the 1737-residue protein in a highly conserved region of the RasGAP-C-terminal (RGCT) domain. When grown at restrictive temperature, strains expressing the sepGG1637R (sepG1) allele are aseptate, with reduced colony growth and aberrantly formed conidiophores. The aseptate condition can be replicated by deletion of AN9463 or by downregulating its expression via introduced promoters. The mutation does not prevent assembly of a cortical contractile actomyosin ring (CAR) at putative septation sites, but tight compaction of the rings is impaired and the rings fail to constrict. Both GFP::SepG wild type and the GFP-tagged product of the sepG1 allele localize to the CAR at both permissive and restrictive temperatures. Downregulation of myoB (encoding the A. nidulans type-II myosin heavy chain) does not prevent formation of SepG rings at septation sites, but filamentous actin is required for CAR localization of SepG and MyoB. We identify fourteen probable IQ-motifs (EF-hand protein binding sites) in the predicted SepG sequence. Two of the A. nidulans EF-hand proteins, myosin essential light chain (AnCdc4) and myosin regulatory light chain (MrlC), colocalize with SepG and MyoB at all stages of CAR formation and constriction. However, calmodulin (CamA) appears at septation sites only after the CAR has become fully compacted. When expression of sepG is downregulated, leaving MyoB as the sole IQ-motif protein in the pre-compaction CAR, both MrlC and AnCdc4 continue to associate with the forming CAR. When myoB expression is downregulated, leaving SepG as the sole IQ-motif protein in the CAR, AnCdc4 association with the forming CAR continues but MrlC fails to associate. This supports a model in which the IQ motifs of MyoB bind both MrlC and AnCdc4, while the IQ motifs of SepG bind only AnCdc4. Downregulation of either mrlC or Ancdc4 results in an aseptate phenotype, but has no effect on association of either SepG or MyoB with the CAR.

Use of enhancer trapping to identify pathogen-induced regulatory events spatially restricted to plant-microbe interaction sites.

Plant genes differentially expressed during plant-pathogen interactions can be important for host immunity or can contribute to pathogen virulence. Large-scale transcript profiling studies, such as microarray- or mRNA-seq-based analyses, have revealed hundreds of genes that are differentially expressed during plant-pathogen interactions. However, transcriptional responses limited to a small number of cells at infection sites can be difficult to detect using these approaches, as they are under-represented in the whole-tissue datasets typically generated by such methods. This study examines the interactions between Arabidopsis thaliana (Arabidopsis) and the pathogenic oomycete Hyaloperonospora arabidopsidis (Hpa) by enhancer trapping to uncover novel plant genes involved in local infection responses. We screened a ß-glucuronidase (GUS) reporter-based enhancer-trap population for expression patterns related to Hpa infection. Several independent lines exhibited GUS expression in leaf mesophyll cells surrounding Hpa structures, indicating a regulatory response to pathogen infection. One of these lines contained a single enhancer-trap insertion in an exon of At1g08800 (MyoB1, Myosin Binding Protein 1) and was subsequently found to exhibit reduced susceptibility to Hpa. Two additional Arabidopsis lines with T-DNA insertions in exons of MyoB1 also exhibited approximately 30% fewer spores than wild-type plants. This study demonstrates that our enhancer-trapping strategy can result in the identification of functionally relevant pathogen-responsive genes. Our results further suggest that MyoB1 either positively contributes to Hpa virulence or negatively affects host immunity against this pathogen.