The Arabidopsis MAP65s are a protein family with similarity to the microtubule-associated proteins PRC1/Ase1p that accumulate in the spindle midzone during late anaphase in mammals and yeast, respectively. Here we investigate the molecular and functional properties of AtMAP65-5 and improve our understanding of AtMAP65-1 properties. We demonstrate that, in vitro, both proteins promote the formation of a planar network of antiparallel microtubules. In vivo, we show that AtMAP65-5 selectively binds the preprophase band and the prophase spindle microtubule during prophase, whereas AtMAP65-1-GFP selectively binds the preprophase band but does not accumulate at the prophase spindle microtubules that coexists within the same cell. At later stages of mitosis, AtMAP65-1 and AtMAP65-5 differentially label the late spindle and phragmoplast. We present evidence for a mode of action for both proteins that involves the binding of monomeric units to microtubules that "zipper up" antiparallel arranged microtubules through the homodimerization of the N-terminal halves when adjacent microtubules encounter. INTRODUCTIONIn higher plant cells, interphase microtubules occur predominantly at the cell cortex as ordered bundles in close association with the plasma membrane. These so-called cortical microtubules (CMTs) play crucial roles in cell morphogenesis (Wasteneys and Fujita, 2006). In rapidly elongating cells, CMTs are linear bundles that are usually transversely oriented relative to the longest cell axis. When cell expansion slows down, the transverse organization is lost and CMTs become oblique (Lloyd, 1994;Dixit and Cyr, 2004). CMTs are highly dynamic and show higher (de)-polymerization rates than interphase mammalian microtubules (Shaw et al., 2003;Dixit et al., 2006). In contrast to animal and yeast microtubules (MTs), CMTs do not emanate from a well-defined nucleating center. Instead, the MT nucleation activity in interphase plant cells mostly occurs at dispersed sites along pre-existing MTs at the cell's cortex (Murata et al., 2005) in a ␥-tubulin-dependent manner (Pastuglia et al., 2006). This MT-dependent nucleation results in branching patterns of CMTs (Murata et al., 2005) that are subsequently resolved into coaligned CMTs. After nucleation, the majority of the MTs are released from their nucleation site, move in the cell cortex by a hybrid treadmilling mechanism leading to polymer interactions (Shaw et al., 2003), and are incorporated into bundles (Dixit and Cyr, 2004). Significantly, the CMT bundles are not anchored and consequently both MT ends are dynamic. Thus, the ordered patterns of CMT arrays are not correlated with the patterns of the CMT nucleation sites (Dixit et al., 2006) and depend on a yet-to-be-discovered mechanism.To accommodate for the transverse network observed in expanding cells, CMTs form bundles. How these bundles are organized is not clear, and for instance, the polarity of CMTs within bundles is not yet solved. Using an in vitro model that provides good access to the cortex combined with the hoo...

Klebsiella pneumoniae is of growing public health concern due to the emergence of strains that are multidrug resistant, virulent, or both. Taxonomically, the K. pneumoniae complex (“Kp”) includes seven phylogroups, with Kp1 (K. pneumoniae sensu stricto) being medically prominent. Kp can be present in environmental sources such as soils and vegetation, which could act as reservoirs of animal and human infections. However, the current lack of screening methods to detect Kp in complex matrices limits research on Kp ecology. Here, we analyzed 1,001 genome sequences and found that existing molecular detection targets lack specificity for Kp. A novel real-time PCR method, the ZKIR (zur-khe intergenic region) assay, was developed and used to detect Kp in 96 environmental samples. The results were compared to a culture-based method using Simmons citrate agar with 1% inositol medium coupled to matrix-assisted laser desorption ionization–time of flight mass spectrometry identification. Whole-genome sequencing of environmental Kp was performed. The ZKIR assay was positive for the 48 tested Kp reference strains, whereas 88 non-Kp strains were negative. The limit of detection of Kp in spiked soil microcosms was 1.5 × 10−1 CFU g−1 after enrichment for 24 h in lysogeny broth supplemented with ampicillin, and it was 1.5 × 103 to 1.5 × 104 CFU g−1 directly after soil DNA extraction. The ZKIR assay was more sensitive than the culture method. Kp was detected in 43% of environmental samples. Genomic analysis of the isolates revealed a predominance of phylogroups Kp1 (65%) and Kp3 (32%), a high genetic diversity (23 multilocus sequence types), a quasi-absence of antibiotic resistance or virulence genes, and a high frequency (50%) of O-antigen type 3. This study shows that the ZKIR assay is an accurate, specific, and sensitive novel method to detect the presence of Kp in complex matrices and indicates that Kp isolates from environmental samples differ from clinical isolates. IMPORTANCE The Klebsiella pneumoniae species complex Kp includes human and animal pathogens, some of which are emerging as hypervirulent and/or antibiotic-resistant strains. These pathogens are diverse and classified into seven phylogroups, which may differ in their reservoirs and epidemiology. Proper management of this public health hazard requires a better understanding of Kp ecology and routes of transmission to humans. So far, detection of these microorganisms in complex matrices such as food or the environment has been difficult due to a lack of accurate and sensitive methods. Here, we describe a novel method based on real-time PCR which enables detection of all Kp phylogroups with high sensitivity and specificity. We used this method to detect Kp isolates from environmental samples, and we show based on genomic sequencing that they differ in antimicrobial resistance and virulence gene content from human clinical Kp isolates. The ZKIR PCR assay will enable rapid screening of multiple samples for Kp presence and will thereby facilitate tracking the dispersal patterns of these pathogenic strains across environmental, food, animal and human sources.