Microtubules (MTs) and actin filaments (F-actin) function cooperatively to regulate plant cell morphogenesis. However, the mechanisms underlying the crosstalk between these two cytoskeletal systems, particularly in cell shape control, remain largely unknown. In this study, we show that introduction of the MyTH4-FERM tandem into KCBP (kinesin-like calmodulin-binding protein) during evolution conferred novel functions. The MyTH4 domain and the FERM domain in the N-terminal tail of KCBP physically bind to MTs and F-actin, respectively. During trichome morphogenesis, KCBP distributes in a specific cortical gradient and concentrates at the branching sites and the apexes of elongating branches, which lack MTs but have cortical F-actin. Further, live-cell imaging and genetic analyses revealed that KCBP acts as a hub integrating MTs and actin filaments to assemble the required cytoskeletal configuration for the unique, polarized diffuse growth pattern during trichome cell morphogenesis. Our findings provide significant insights into the mechanisms underlying cytoskeletal regulation of cell shape determination.DOI: http://dx.doi.org/10.7554/eLife.09351.001

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Background: Cotton Verticillium wilt is one of the most devastating diseases for cotton production in the world. Although this diseases have been widely studied at the molecular level from pathogens, the molecular basis of V. dahliae interacted with cotton has not been well examined. Results: In this study, RNA-seq analysis was carried out on V. dahliae samples cultured by different root exudates from three cotton cultivars (a susceptible upland cotton cultivar, a tolerant upland cotton cultivar and a resistant island cotton cultivar) and water for 0 h, 6 h, 12 h, 24 h and 48 h. Statistical analysis of differentially expressed genes revealed that V. dahliae responded to all kinds of root exudates but more strongly to susceptible cultivar than to tolerant and resistant cultivars. Go analysis indicated that 'hydrolase activity, hydrolyzing O-glycosyl compounds' related genes were highly enriched in V. dahliae cultured by root exudates from susceptible cotton at early stage of interaction, suggesting genes related to this term were closely related to the pathogenicity of V. dahliae. Additionally, 'transmembrane transport', 'coenzyme binding', 'NADP binding', 'cofactor binding', 'oxidoreductase activity', 'flavin adenine dinucleotide binding', 'extracellular region' were commonly enriched in V. dahliae cultured by all kinds of root exudates at early stage of interaction (6 h and 12 h), suggesting that genes related to these terms were required for the initial steps of the roots infections. Conclusions: Based on the GO analysis results, the early stage of interaction (6 h and 12 h) were considered as the critical stage of V. dahliae-cotton interaction. Comparative transcriptomic analysis detected that 31 candidate genes response to root exudates from cotton cultivars with different level of V. dahliae resistance, 68 response to only susceptible cotton cultivar, and 26 genes required for development of V. dahliae. Collectively, these expression data have advanced our understanding of key molecular events in the V. dahliae interacted with cotton, and provided a framework for further functional studies of candidate genes to develop better control strategies for the cotton wilt disease.

Fibre cell initiation and elongation is critical for cotton fibre development. However, little is known about the regulation of initiation and elongation during fibre cell development. Here, the regulatory role of a novel protein GhCFE1A was uncovered. GhCFE1A is preferentially expressed at initiation and rapid elongation stages during fibre development; in addition, much higher expression of GhCFE1A was detected at the fibre initiation stage in fibreless cotton mutants than in the fibre-bearing TM-1 wild-type. Importantly, overexpression of GhCFE1A in cotton not only delayed fibre cell elongation but also significantly reduced the density of lint and fuzz fibre initials and stem trichomes. Yeast two-hybrid assay showed that GhCFE1A interacted with several actin proteins, and the interaction was further confirmed by co-sedimentation assay. Interestingly, a subcellular localization assay showed that GhCFE1A resided on the cortical endoplasmic reticulum (ER) network and co-localized with actin cables. Moreover, the density of F-actin filaments was shown to be reduced in GhCFE1A-overexpressing fibres at the rapid elongation stage compared with the wild-type control. Taken together, the results demonstrate that GhCFE1A probably functions as a dynamic linker between the actin cytoskeleton and the ER network, and plays an important role in fibre cell initiation and elongation during cotton fibre development.

The vascular wilt fungus Verticillium dahliae is one of the most destructive pathogens of cotton (Gossypium hirsutum) and many other economically important dicot plants. Fungal pathogens require Myosin-mediated actomyosin motility system for colonization of their host plants; however, the mechanisms underlying this process have not been fully characterized for V. dahliae. Here, in a knock-out experiment, we characterized the role of VdMyo5, a member of the Myosin V family, before and during infection of cotton and Arabidopsis thaliana. The VdMyo5 deletion mutant (ΔVdmyo5) fungi showed obvious defects in the development of conidia and the polarized elongation of vegetative hyphae, but no inhibition of host root penetration. Overall, the ΔVdmyo5 fungi exhibited dramatically reduced virulence in cotton and Arabidopsis, with almost no colonization in sections of host vascular tissue. We found labelled Myosin5-GFP to be specifically enriched at the hyphal tip, co-localized with FM4-64 labelled Spitzenkörper, which is the vesicle supply centre in filamentous fungi. Comparative secretome analysis revealed that proteins associated with cell wall modification and degradation of reactive oxygen species were significantly altered in mutant strains. Our results indicate that Myosin5 is required for vegetative growth and full virulence, possibly by regulating vesicle transport. The findings provide important insight into the cellular mechanisms of Verticillium pathogenesis.

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