Visualization of Highly Dynamic F-Actin Plus Ends in Growing Phaseolus vulgaris Root Hair Cells and Their Responses to Rhizobium etli Nod Factors

Abstract

Legume plants secrete signaling molecules called flavonoids into the rhizosphere. These molecules activate the transcription of rhizobial nod genes, which encode proteins involved in the synthesis of signaling compounds named Nod factors (NFs). NFs, in turn, trigger changes in plant gene expression, cortical cell dedifferentiation and mitosis, depolarization of the root hair cell membrane potential and rearrangement of the actin cytoskeleton. Actin polymerization plays an important role in apical growth in hyphae and pollen tubes. Using sublethal concentrations of fluorescently labeled cytochalasin D (Cyt-Fl), we visualized the distribution of filamentous actin (F-actin) plus ends in living Phaseolus vulgaris and Arabidopsis root hairs during apical growth. We demonstrated that Cyt-Fl specifically labeled the newly available plus ends of actin microfilaments, which probably represent sites of polymerization. The addition of unlabeled competing cytochalasin reduced the signal, suggesting that the labeled and unlabeled forms of the drug bind to the same site on F-actin. Exposure to Rhizobium etli NFs resulted in a rapid increase in the number of F-actin plus ends in P. vulgaris root hairs and in the re-localization of F-actin plus ends to infection thread initiation sites. These data suggest that NFs promote the formation of F-actin plus ends, which results in actin cytoskeleton rearrangements that facilitate infection thread formation.

Biotechnological aspects of cytoskeletal regulation in plants

“…Nowadays, microscopic approaches to study cytoskeleton dynamics and reorganization have been improved , and thus they may allow deeper insight into the host/ microbe interactions in space and time (Perrine-Walker et al, 2014;Zepeda et al, 2014). In the following sections, the host cytoskeletal responses upon symbiotic or pathogenic plant-microbe interactions will be examined.…”

“…Mutations in NAP1 and PIR1 genes showed that these SCAR/WAVE complex proteins are essential for root hair, trichome and infection thread growth (Miyahara et al, 2010). Recently, a quantitative assessment of actin microfilament dynamics during apical growth was carried out using substoichiometric concentrations of fluorescently labeled cytochalasin D below its IC50 (concentration of cytochalasin D exhibiting half-maximal inhibitory activity against actin microfilament polymerization) which purportedly marks barbed ends of filamentous actin where actin nucleation occurs (Zepeda et al, 2014). After exposure to Nod factor, number of plus actin ends significantly increased in Phaseolus vulgaris root hairs and these plus ends relocalized to the site of infection thread.…”

Actin polymerization drives polar growth inArabidopsisroot hair cells

“…9 In this work, we examined the distribution of polymerizing actin using confocal microscopy. Although the fluorescently tagged plus ends were mainly localized in the tip region, some actin foci were distributed in the cytoplasm.…”

“…Furthermore, using a low concentration of fluorescently labeled cytochalasin, we recently reported that free actin plus ends (i.e., growing or polymerizing ends) are located in the tip region of growing root hairs. 9 Under such low, sublethal concentrations, the intensity of the actin polymerization signal in the tip increased in growing root hair cells treated with Rhizobium etli nodulation factors (NFs), declined during the swelling response, and was restored to normal levels when tip growth was reinitiated. This finding adds weight to the idea that actin polymerization plays a key role in the tip region of growing root hair cells.…”

“…Importantly, we found that the signal was strongest in close proximity to the plasma membrane in the tip dome, and exhibited dynamics that seemed to be correlated with tip growth. The visualization of actin plus ends using a sublethal concentration of fluorescently labeled cytochalasin Arabidopsis root hairs were treated with a sublethal concentration of fluorescently labeled cytochalasin as reported 9 and observed at low magnification (objective10X) under a confocal microscope. As reported previously, most of the fluorescence signal was focused in the tip region, and the root hairs continued to grow normally, as judged by the continuation of cytoplasmic streaming and robust growth rate.…”