No abstract

Antimicrobial susceptibility patterns of 112 clinical isolates, 28 type strains, and 9 reference strains of Nocardia were determined using the Sensititre Rapmyco microdilution panel (Thermo Fisher, Inc.). Isolates were identified by highly discriminatory multilocus sequence analysis and were chosen to represent the diversity of species recovered from clinical specimens in Ontario, Canada. Susceptibility to the most commonly used drug, trimethoprim-sulfamethoxazole, was observed in 97% of isolates. Linezolid and amikacin were also highly effective; 100% and 99% of all isolates demonstrated a susceptible phenotype. For the remaining antimicrobials, resistance was species specific with isolates of Nocardia otitidiscaviarum, N. brasiliensis, N. abscessus complex, N. nova complex, N. transvalensis complex, N. farcinica, and N. cyriacigeorgica displaying the traditional characteristic drug pattern types. In addition, the antimicrobial susceptibility profiles of a variety of rarely encountered species isolated from clinical specimens are reported for the first time and were categorized into four additional drug pattern types. N ocardia species are a group of filamentous, branching, Grampositive, modified-acid-fast bacilli that normally exist as soil saprophytes but can cause disease in immunosuppressed and healthy individuals (1). Most infections involve inhalation of fragments of filaments, resulting in pulmonary nocardiosis and pneumonia, which can be followed by dissemination to the heart, skin, subcutaneous tissue, and central nervous system (1).Nocardia taxonomy has been linked to specific patterns of antimicrobial susceptibility ever since the foundational work by Wallace et al. (2) (2, 4). With the clinical application of DNA sequencing, Nocardia taxonomy has changed and expanded rapidly. Previously identified species have been reclassified as species complexes encompassing multiple species, and numerous novel Nocardia species have been identified (5). Currently, 87 species are enumerated in the List of Prokaryotic Names with Standing in Nomenclature (LPSN) (http://www.bacterio.net/nocardia.html), many of which are clinically significant (1).However, data on antimicrobial susceptibility has lagged behind the advances in taxonomy, with only a few reports providing recent data on newer antimicrobials (4,6,7,8,9,10,11,12,13). These reports largely focus on the species traditionally associated with clinical infections, such that antimicrobial susceptibility data are not available for a large number of newly identified, but clinically relevant, Nocardia species that are isolated less frequently in the clinical laboratory. Furthermore, the species in these studies were identified using microscopy and biochemical testing, or the identification relied heavily on the 16S rRNA gene sequence analysis, both of which are unable to reliably discriminate many Nocardia species (5,14,15).The purpose of this study was to profile the antimicrobial susceptibility patterns of a diverse range of Nocardia species isolated from clinica...

Author contribution statement JD and DRG designed the research. AD, ZG and MKN provided unpublished stigmatic papillae transcriptome datasets for further analysis. JD, CT, EFW and HKL performed the research. JD and DRG conducted the data analysis, prepared the figures and wrote the manuscript. JD, DRG and HKL edited the manuscript. We are very grateful to Alexander Leydon (Mark Johnson lab, Brown University) for providing valuable advice on using the CRISPR/Cas9 system, and Audrey Darabie for assistance with the SEM. We acknowledge the Salk Institute Genomic Analysis Laboratory (SIGnAL), GABI-Kat, and the Arabidopsis Biological Resource Center (ABRC) for providing the sequence-indexed Arabidopsis T-DNA insertion mutant, and Addgene and ABRC for providing the CRISPR/Cas9 vectors. We also thank Betty Geng for technical assistance with genotyping plants, and members of the Goring lab for critically reading this manuscript.

BackgroundIn the Brassicaceae, the early stages of compatible pollen-stigma interactions are tightly controlled with early checkpoints regulating pollen adhesion, hydration and germination, and pollen tube entry into the stigmatic surface. However, the early signalling events in the stigma which trigger these compatible interactions remain unknown.ResultsA set of stigma-expressed pseudokinase genes, termed BRASSIKINs (BKNs), were identified and found to be present in only core Brassicaceae genomes. In Arabidopsis thaliana Col-0, BKN1 displayed stigma-specific expression while the BKN2 gene was expressed in other tissues as well. CRISPR deletion mutations were generated for the two tandemly linked BKNs, and very mild hydration defects were observed for wild-type Col-0 pollen when placed on the bkn1/2 mutant stigmas. In further analyses, the predominant transcript for the stigma-specific BKN1 was found to have a premature stop codon in the Col-0 ecotype, but a survey of the 1001 Arabidopsis genomes uncovered three ecotypes that encoded a full-length BKN1 protein. Furthermore, phylogenetic analyses identified intact BKN1 orthologues in the closely related outcrossing Arabidopsis species, A. lyrata and A. halleri. Finally, the BKN pseudokinases were found to be plasma-membrane localized through the dual lipid modification of myristoylation and palmitoylation, and this localization would be consistent with a role in signaling complexes.ConclusionIn this study, we have characterized the novel Brassicaceae-specific family of BKN pseudokinase genes, and examined the function of BKN1 and BKN2 in the context of pollen-stigma interactions in A. thaliana Col-0. Additionally, premature stop codons were identified in the predicted stigma specific BKN1 gene in a number of the 1001 A. thaliana ecotype genomes, and this was in contrast to the out-crossing Arabidopsis species which carried intact copies of BKN1. Thus, understanding the function of BKN1 in other Brassicaceae species will be a key direction for future studies.