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Hydrogen peroxide (H2O2) in plants is produced in relatively large amounts and plays a universal role in plant defense and physiological responses, including the regulation of growth and development. In the Rhizobium–legume symbiosis, hydrogen peroxide plays an important signaling role throughout the development of this interaction. In the functioning nodule, H2O2 has been shown to be involved in bacterial differentiation into the symbiotic form and in nodule senescence. In this study, the pattern of H2O2 accumulation in pea (Pisum sativum L.) wild-type and mutant nodules blocked at different stages of the infection process was analyzed using a cytochemical reaction with cerium chloride. The observed dynamics of H2O2 deposition in the infection thread walls indicated that the distribution of H2O2 was apparently related to the stiffness of the infection thread wall. The dynamics of H2O2 accumulation was traced, and its patterns in different nodule zones were determined in order to investigate the relationship of H2O2 localization and distribution with the stages of symbiotic nodule development in P. sativum. The patterns of H2O2 localization in different zones of the indeterminate nodule have been partially confirmed by comparative analysis on mutant genotypes. [ABSTRACT FROM AUTHOR]

Most legumes can form an endosymbiotic association with soil bacteria called rhizobia, which colonize specialized root structures called nodules where they fix nitrogen. To colonize nodule cells, rhizobia must first traverse the epidermis and outer cortical cell layers of the root. In most legumes, this involves formation of the infection thread, an intracellular structure that becomes colonized by rhizobia, guiding their passage through the outer cell layers of the root and into the newly formed nodule cells. In this brief review, we recount the early research milestones relating to the rhizobial infection thread and highlight two relatively recent advances in the symbiotic infection mechanism, the eukaryotically conserved 'MYB–AUR1–MAP' mitotic module, which links cytokinesis mechanisms to intracellular infection, and the discovery of the 'infectosome' complex, which guides infection thread growth. We also discuss the potential intertwining of the two modules and the hypothesis that cytokinesis served as a foundation for intracellular infection of symbiotic microbes. [ABSTRACT FROM AUTHOR]

Hydrogen peroxide (H2O2) in plants is produced in relatively large amounts and plays a universal role in plant defense and physiological responses, including the regulation of growth and development. In the Rhizobium–legume symbiosis, hydrogen peroxide plays an important signaling role throughout the development of this interaction. In the functioning nodule, H2O2 has been shown to be involved in bacterial differentiation into the symbiotic form and in nodule senescence. In this study, the pattern of H2O2 accumulation in pea (Pisum sativum L.) wild-type and mutant nodules blocked at different stages of the infection process was analyzed using a cytochemical reaction with cerium chloride. The observed dynamics of H2O2 deposition in the infection thread walls indicated that the distribution of H2O2 was apparently related to the stiffness of the infection thread wall. The dynamics of H2O2 accumulation was traced, and its patterns in different nodule zones were determined in order to investigate the relationship of H2O2 localization and distribution with the stages of symbiotic nodule development in P. sativum. The patterns of H2O2 localization in different zones of the indeterminate nodule have been partially confirmed by comparative analysis on mutant genotypes.

ABSTRACT Bacterial chromosome segregation is facilitated by the ParABS system. The ParB protein binds centromere-like parS sequences and forms nucleoprotein complexes. These nucleoprotein complexes are segregated by the dynamic ATPase ParA. In mycobacteria, ParA also interacts with the polar growth determinant DivIVA (Wag31). This interaction was earlier shown not only to facilitate the segregation of ParB complexes but also to affect cell extension. Here, we identify an additional partner of ParA in Mycobacterium smegmatis, named PapM. Using E. coli-based analysis, we show that PapM likewise interacts with DivIVA and that the tripartite interaction of ParA-PapM-DivIVA is phosphorylation dependent: ParA binding to DivIVA is diminished, while PapM binding is promoted upon phosphorylation of DivIVA. The presence of PapM enhances the dissociation of ParA from the DivIVA complex upon its phosphorylation. Studies of M. smegmatis mutant strains reveal that altered PapM levels influence chromosome segregation and cell length. The elimination of PapM affects ParA dynamics. Furthermore, ParA and, to a lesser extent, PapM modulate the subcellular distribution of DivIVA. Altogether, our studies show that the tripartite interplay between ParA-DivIVA and PapM controls the switch between cell division and cell elongation and, in this way, affects the mycobacterial cell cycle. IMPORTANCE The genus of Mycobacterium includes important clinical pathogens (M. tuberculosis). Bacteria of this genus share the unusual features of their cell cycle such as asymmetric polar cell elongation and long generation time. Markedly, control of the mycobacterial cell cycle still remains not fully understood. The main cell growth determinant in mycobacteria is the essential protein DivIVA, which is also involved in cell division. DivIVA activity is controlled by phosphorylation, but the mechanism and significance of this process are unknown. Here, we show how the previously established protein interaction partner of DivIVA in mycobacteria, the segregation protein ParA, affects the DivIVA subcellular distribution. We also demonstrate the role of a newly identified M. smegmatis DivIVA and ParA interaction partner, a protein named PapM, and we establish how their interactions are modulated by phosphorylation. Demonstrating that the tripartite interplay affects the mycobacterial cell cycle contributes to the general understanding of mycobacterial growth regulation.

Agrobacterium tumefaciens C58 contains four replicons, circular chromosome (CC), linear chromosome (LC), cryptic plasmid (pAt), and tumor-inducing plasmid (pTi), and grows by polar growth from a single growth pole (GP), while the old cell compartment and its old pole (OP) do not elongate. We monitored the replication and segregation of these four genetic elements during polar growth. The three largest replicons (CC, LC, pAt) reside in the OP compartment prior to replication; post replication one copy migrates to the GP prior to division. CC resides at a fixed location at the OP and replicates first. LC does not stay fixed at the OP once the cell cycle begins and replicates from varied locations 20 min later than CC. pAt localizes similarly to LC prior to replication, but replicates before the LC and after the CC. pTi does not have a fixed location, and post replication it segregates randomly throughout old and new cell compartments, while undergoing one to three rounds of replication during a single cell cycle. Segregation of the CC and LC is dependent on the GP and OP identity factors PopZ and PodJ, respectively. Without PopZ, replicated CC and LC do not efficiently partition, resulting in sibling cells without CC or LC. Without PodJ, the CC and LC exhibit abnormal localization to the GP at the beginning of the cell cycle and replicate from this position. These data reveal PodJ plays an essential role in CC and LC tethering to the OP during early stages of polar growth.

Agrobacterium tumefaciens grows by addition of peptidoglycan (PG) at one pole of the bacterium. During the cell cycle, the cell needs to maintain two different developmental programs, one at the growth pole and another at the inert old pole. Proteins involved in this process are not yet well characterized. To further characterize the role of pole-organizing protein A. tumefaciens PopZ (PopZ At ), we created deletions of the five PopZ At domains and assayed their localization. In addition, we created a popZAt deletion strain (ΔpopZAt ) that exhibited growth and cell division defects with ectopic growth poles and minicells, but the strain is unstable. To overcome the genetic instability, we created an inducible PopZ At strain by replacing the native ribosome binding site with a riboswitch. Cultivated in a medium without the inducer theophylline, the cells look like ΔpopZAt cells, with a branching and minicell phenotype. Adding theophylline restores the wild-type (WT) cell shape. Localization experiments in the depleted strain showed that the domain enriched in proline, aspartate, and glutamate likely functions in growth pole targeting. Helical domains H3 and H4 together also mediate polar localization, but only in the presence of the WT protein, suggesting that the H3 and H4 domains multimerize with WT PopZ At , to stabilize growth pole accumulation of PopZ AtIMPORTANCEAgrobacterium tumefaciens is a rod-shaped bacterium that grows by addition of PG at only one pole. The factors involved in maintaining cell asymmetry during the cell cycle with an inert old pole and a growing new pole are not well understood. Here we investigate the role of PopZ At , a homologue of Caulobacter crescentus PopZ (PopZ Cc ), a protein essential in many aspects of pole identity in C. crescentus We report that the loss of PopZ At leads to the appearance of branching cells, minicells, and overall growth defects. As many plant and animal pathogens also employ polar growth, understanding this process in A. tumefaciens may lead to the development of new strategies to prevent the proliferation of these pathogens. In addition, studies of A. tumefaciens will provide new insights into the evolution of the genetic networks that regulate bacterial polar growth and cell division.

Rho1p is a membrane-associated protein that belongs to the Rho family of small GTPases. These proteins coordinate processes such as actin remodelling and polarised secretion to maintain the shape and homeostasis of yeast cells. In response to extracellular stimuli, Rho1p undergoes conformational switching between a guanosine triphosphate (GTP)-bound active state and a guanosine diphosphate (GDP)-bound inactive state. Cycling is improved with guanine nucleotide exchange factor (GEF) activity necessary to activate signalling and GTPase activating protein (GAP) activity required for subsequent signal depletion. This review focuses on fission yeast Rho1p GEFs, Rgf1p, Rgf2p, and Rgf3p that belong to the family of DH-PH domain-containing Dbl-related GEFs. They are multi-domain proteins that detect biological signals that induce or inhibit their catalytic activity over Rho1p. Each of them activates Rho1p in different places and times. Rgf1p acts preferentially during polarised growth. Rgf2p is required for sporulation, and Rgf3p plays an essential function in septum synthesis. In addition, we outline the noncanonical roles of Rho1p-GEFs in genomic instability. [ABSTRACT FROM AUTHOR]

Mycobacteria, including the human pathogen Mycobacterium tuberculosis, grow by inserting new cell wall material at their poles. This process and that of division are asymmetric, producing a phenotypically heterogeneous population of cells that respond non-uniformly to stress (Aldridge et al., 2012; Rego et al., 2017). Surprisingly, deletion of a single gene – lamA – leads to more symmetry, and to a population of cells that is more uniformly killed by antibiotics (Rego et al., 2017). How does LamA create asymmetry? Here, using a combination of quantitative time-lapse imaging, bacterial genetics, and lipid profiling, we find that LamA recruits essential proteins involved in cell wall synthesis to one side of the cell – the old pole. One of these proteins, MSMEG_0317, here renamed PgfA, was of unknown function. We show that PgfA is a periplasmic protein that interacts with MmpL3, an essential transporter that flips mycolic acids in the form of trehalose monomycolate (TMM), across the plasma membrane. PgfA interacts with a TMM analog suggesting a direct role in TMM transport. Yet our data point to a broader function as well, as cells with altered PgfA levels have differences in the abundance of other lipids and are differentially reliant on those lipids for survival. Overexpression of PgfA, but not MmpL3, restores growth at the old poles in cells missing lamA. Together, our results suggest that PgfA is a key determinant of polar growth and cell envelope composition in mycobacteria, and that the LamA-mediated recruitment of this protein to one side of the cell is a required step in the establishment of cellular asymmetry.

Root hairs are tubular outgrowths of epidermal cells that increase the root surface area and thereby make the root more efficient at absorbing water and nutrients. Their expansion is limited to the root hair apex, where growth is reported to take place in a pulsating manner. These growth pulses coincide with oscillations of the apoplastic and cytosolic pH in a similar way as has been reported for pollen tubes. Likewise, the concentrations of apoplastic reactive oxygen species (ROS) and cytoplasmic Ca2+ oscillate with the same periodicity as growth. Whereas ROS appear to control cell wall extensibility and opening of Ca2+ channels, the role of protons as a growth signal in root hairs is less clear andmay differ fromthat in pollen tubes where plasmamembrane H+-ATPases have been shown to sustain growth. In this review, we outline our current understanding of how pH contributes to root hair development. [ABSTRACT FROM AUTHOR]

Abstract Filamentous fungal cell factories are efficient producers of platform chemicals, proteins, enzymes and natural products. Stirred-tank bioreactors up to a scale of several hundred m³ are commonly used for their cultivation. Fungal hyphae self-assemble into various cellular macromorphologies ranging from dispersed mycelia, loose clumps, to compact pellets. Development of these macromorphologies is so far unpredictable but strongly impacts productivities of fungal bioprocesses. Depending on the strain and the desired product, the morphological forms vary, but no strain- or product-related correlations currently exist to improve process understanding of fungal production systems. However, novel genomic, genetic, metabolic, imaging and modelling tools have recently been established that will provide fundamental new insights into filamentous fungal growth and how it is balanced with product formation. In this primer, these tools will be highlighted and their revolutionary impact on rational morphology engineering and bioprocess control will be discussed.

Agrobacterium tumefaciens grows by addition of peptidoglycan (PG) at one pole of the bacterium. During the cell cycle, the cell needs to maintain two different developmental programs, one at the growth pole and another at the inert old pole. Proteins involved in this process are not yet well characterized. To further characterize the role of pole-organizing protein A. tumefaciens PopZ (PopZ At ), we created deletions of the five PopZ At domains and assayed their localization. In addition, we created a popZAt deletion strain (ΔpopZAt ) that exhibited growth and cell division defects with ectopic growth poles and minicells, but the strain is unstable. To overcome the genetic instability, we created an inducible PopZ At strain by replacing the native ribosome binding site with a riboswitch. Cultivated in a medium without the inducer theophylline, the cells look like ΔpopZAt cells, with a branching and minicell phenotype. Adding theophylline restores the wild-type (WT) cell shape. Localization experiments in the depleted strain showed that the domain enriched in proline, aspartate, and glutamate likely functions in growth pole targeting. Helical domains H3 and H4 together also mediate polar localization, but only in the presence of the WT protein, suggesting that the H3 and H4 domains multimerize with WT PopZ At , to stabilize growth pole accumulation of PopZ AtIMPORTANCEAgrobacterium tumefaciens is a rod-shaped bacterium that grows by addition of PG at only one pole. The factors involved in maintaining cell asymmetry during the cell cycle with an inert old pole and a growing new pole are not well understood. Here we investigate the role of PopZ At , a homologue of Caulobacter crescentus PopZ (PopZ Cc ), a protein essential in many aspects of pole identity in C. crescentus We report that the loss of PopZ At leads to the appearance of branching cells, minicells, and overall growth defects. As many plant and animal pathogens also employ polar growth, understanding this process in A. tumefaciens may lead to the development of new strategies to prevent the proliferation of these pathogens. In addition, studies of A. tumefaciens will provide new insights into the evolution of the genetic networks that regulate bacterial polar growth and cell division.

Root hairs are tubular outgrowths of epidermal cells that increase the root surface area and thereby make the root more efficient at absorbing water and nutrients. Their expansion is limited to the root hair apex, where growth is reported to take place in a pulsating manner. These growth pulses coincide with oscillations of the apoplastic and cytosolic pH in a similar way as has been reported for pollen tubes. Likewise, the concentrations of apoplastic reactive oxygen species (ROS) and cytoplasmic Ca2+ oscillate with the same periodicity as growth. Whereas ROS appear to control cell wall extensibility and opening of Ca2+ channels, the role of protons as a growth signal in root hairs is less clear and may differ from that in pollen tubes where plasma membrane H+-ATPases have been shown to sustain growth. In this review, we outline our current understanding of how pH contributes to root hair development.

Phytophthora capsici is a destructive plant pathogen that infects a wide range of hosts worldwide. The P. capsici cell wall, rich in cellulose, is vital for hyphal growth and host interactions. However, the enzymes involved in its synthesis remain largely unelucidated. In the current study, we functionally characterized the cellulose synthase gene PcCesA1 , which is highly conserved in Phytophthora. By using CRISPR/Cas9-mediated gene replacement and in situ complementation system, it was found PcCesA1 is essential for the mycelial growth, cystospore germination, and pathogenicity of P. capsici. The normal deposition of newly synthesized cell wall components and the polar growth point formation were disrupted in PcCesA1 knockout mutants, suggesting that PcCesA1 plays an important role in the polar growth of P. capsici. Compared with the wild-type strains, PcCesA1 knockout mutants displayed a thicker inner layer cell wall and were more sensitive to carboxylic acid amide fungicides (CAAs). The contents of the cell wall polysaccharides 1,4-Glc, 1,4,6-Glc, and 1,3,4-Glc were reduced in PcCesA1 knockout mutants, suggesting that PcCesA1 affected cellulose content and glycosidic linkage crosslinking in the cell wall. Our findings demonstrate that PcCesA1 is required for cell wall biogenesis. Therefore, PcCesA1 may be a potential target for Phytophthora disease control. [ABSTRACT FROM AUTHOR]

Unlike other bacteria, cell growth in rhizobiales is unipolar and asymmetric. The regulation of cell division, and its coordination with metabolic processes is an active field of research. In Rhizobium etli , gene RHE_PE00024, located in a secondary chromosome, is essential for growth. This gene encodes a predicted hybrid histidine kinase sensor protein, participating in a, as yet undescribed, two-component signaling system. In this work, we show that a conditional knockdown mutant (cKD24) in RHE_PE00024 (hereby referred as rdsA , after rhizobium division and shape) generates a striking phenotype, where nearly 64% of the cells present a round shape, with stochastic and uncoordinated cell division. For rod-shaped cells, a large fraction (12 to 29%, depending on their origin) present growth from the old pole, a sector that is normally inactive for growth in a wild-type cell. A fraction of the cells (1 to 3%) showed also multiple ectopic polar growths. Homodimerization of RdsA appears to be required for normal function. RNAseq analysis of mutant cKD24 reveals global changes, with downregulated genes in at least five biological processes: cell division, wall biogenesis, respiration, translation, and motility. These modifications may affect proper structuring of the divisome, as well as peptidoglycan synthesis. Together, these results indicate that the hybrid histidine kinase RdsA is an essential global regulator influencing cell division and cell shape in R. etli. [ABSTRACT FROM AUTHOR]

Unlike other bacteria, cell growth in rhizobiales is unipolar and asymmetric. The regulation of cell division, and its coordination with metabolic processes is an active field of research. In Rhizobium etli, gene RHE_PE00024, located in a secondary chromosome, is essential for growth. This gene encodes a predicted hybrid histidine kinase sensor protein, participating in a, as yet undescribed, two-component signaling system. In this work, we show that a conditional knockdown mutant (cKD24) in RHE_PE00024 (hereby referred as rdsA, after rhizobium division and shape) generates a striking phenotype, where nearly 64% of the cells present a round shape, with stochastic and uncoordinated cell division. For rod-shaped cells, a large fraction (12 to 29%, depending on their origin) present growth from the old pole, a sector that is normally inactive for growth in a wild-type cell. A fraction of the cells (1 to 3%) showed also multiple ectopic polar growths. Homodimerization of RdsA appears to be required for normal function. RNAseq analysis of mutant cKD24 reveals global changes, with downregulated genes in at least five biological processes: cell division, wall biogenesis, respiration, translation, and motility. These modifications may affect proper structuring of the divisome, as well as peptidoglycan synthesis. Together, these results indicate that the hybrid histidine kinase RdsA is an essential global regulator influencing cell division and cell shape in R. etli.

Pyriofenone demonstrates outstanding efficacy in controlling powdery mildew. We investigated the impact of pyriofenone on the infection processes and cytological features of Blumeria graminis f. sp. tritici on wheat leaves. The preventive application of pyriofenone before inoculation did not inhibit conidial germination but effectively suppressed both appressorial and haustorial formation. Notably, haustorial formation was effectively inhibited, resulting in the complete suppression of successive lesion development and sporulation. Curative application of pyriofenone after inoculation also inhibited lesion expansion and sporulation. Furthermore, it had considerable impact on the morphogenesis of powdery mildew fungus. We observed multi-formed secondary appressoria, shrunken or bifurcated hyphae, abnormal conidiophores, and clubbed conidia-like structures. Subsequently, we employed a histochemical approach to analyze the localization of essential components for the polar growth of fungal hyphae. Pyriofenone induced mislocalization of the actin cytoskeleton, β-glucan and cytoplasmic vesicles, although it did not affect tubulin orientation., (© 2024 Pesticide Science Society of Japan.)

Proline‐rich extensin‐like receptor kinases (PERKs) belong to the hydroxyproline‐rich glycoprotein (HRGP) superfamily known to be involved in many plant developmental processes. Here, we characterized two pollen‐expressed PERKs from Arabidopsis thaliana, PERK5 and PERK12. Pollen tube growth was impaired in single and double perk5‐1 perk12‐1 loss of function mutants, with an impact on seed production. When the segregation was analysed, a male gametophytic defect was found, indicating that perk5‐1 and perk12‐1 mutants carry deficient pollen transmission. Furthermore, perk5‐1 perk12‐1 displayed an excessive accumulation of pectins and cellulose at the cell wall of the pollen tubes. Our results indicate that PERK5 and PERK12 are necessary for proper pollen tube growth, highlighting their role in cell wall assembly and reactive oxygen species homeostasis. [ABSTRACT FROM AUTHOR]

Agrobacterium tumefaciens elongates by addition of peptidoglycan (PG) only at the pole created by cell division, the growth pole, whereas the opposite pole, the old pole, is inactive for PG synthesis. How Agrobacterium assigns and maintains pole asymmetry is not understood. Here, we investigated whether polar growth is correlated with novel pole-specific localization of proteins implicated in a variety of growth and cell division pathways. The cell cycle of A. tumefaciens was monitored by time-lapse and superresolution microscopy to image the localization of A. tumefaciens homologs of proteins involved in cell division, PG synthesis and pole identity. FtsZ and FtsA accumulate at the growth pole during elongation, and improved imaging reveals FtsZ disappears from the growth pole and accumulates at the midcell before FtsA. The L,D-transpeptidase Atu0845 was detected mainly at the growth pole. A. tumefaciens specific pole-organizing protein (Pop) PopZAt and polar organelle development (Pod) protein PodJAt exhibited dynamic yet distinct behavior. PopZAt was found exclusively at the growing pole and quickly switches to the new growth poles of both siblings immediately after septation. PodJAt is initially at the old pole but then also accumulates at the growth pole as the cell cycle progresses suggesting that PodJAt may mediate the transition of the growth pole to an old pole. Thus, PopZAt is a marker for growth pole identity, whereas PodJAt identifies the old pole.

Rho1p is a membrane-associated protein that belongs to the Rho family of small GTPases. These proteins coordinate processes such as actin remodelling and polarised secretion to maintain the shape and homeostasis of yeast cells. In response to extracellular stimuli, Rho1p undergoes conformational switching between a guanosine triphosphate (GTP)-bound active state and a guanosine diphosphate (GDP)-bound inactive state. Cycling is improved with guanine nucleotide exchange factor (GEF) activity necessary to activate signalling and GTPase activating protein (GAP) activity required for subsequent signal depletion. This review focuses on fission yeast Rho1p GEFs, Rgf1p, Rgf2p, and Rgf3p that belong to the family of DH-PH domain-containing Dbl-related GEFs. They are multi-domain proteins that detect biological signals that induce or inhibit their catalytic activity over Rho1p. Each of them activates Rho1p in different places and times. Rgf1p acts preferentially during polarised growth. Rgf2p is required for sporulation, and Rgf3p plays an essential function in septum synthesis. In addition, we outline the noncanonical roles of Rho1p-GEFs in genomic instability.

LytM-domain containing proteins are LAS peptidases (lysostaphin-type enzymes, D-Ala-D-Ala metallopeptidases, and sonic hedgehog) and are known to play diverse roles throughout the bacterial cell cycle through direct or indirect hydrolysis of the bacterial cell wall. A subset of the LytM factors are catalytically inactive but regulate the activity of other cell wall hydrolases and are classically described as cell separation factors NlpD and EnvC. Here, we explore the function of four LytM factors in the alphaproteobacterial plant pathogen Agrobacterium tumefaciens. An LmdC ortholog (Atu1832) and a MepM ortholog (Atu4178) are predicted to be catalytically active. While Atu1832 does not have an obvious function in cell growth or division, Atu4178 is essential for polar growth and likely functions as a space-making endopeptidase that cleaves amide bonds in the peptidoglycan cell wall during elongation. The remaining LytM factors are degenerate EnvC and NlpD orthologs. Absence of these proteins results in striking phenotypes indicative of misregulation of cell division and growth pole establishment. The deletion of an amidase, AmiC, closely phenocopies the deletion of envC suggesting that EnvC might regulate AmiC activity. The NlpD ortholog DipM is unprecedently essential for viability and depletion results in the misregulation of early stages of cell division, contrasting with the canonical view of DipM as a cell separation factor. Finally, we make the surprising observation that absence of AmiC relieves the toxicity induced by dipM overexpression. Together, these results suggest EnvC and DipM may function as regulatory hubs with multiple partners to promote proper cell division and establishment of polarity.

LytM-domain containing proteins are LAS peptidases (lysostaphin-type enzymes, D-Ala-D-Ala metallopeptidases, and sonic hedgehog) and are known to play diverse roles throughout the bacterial cell cycle through direct or indirect hydrolysis of the bacterial cell wall. A subset of the LytM factors are catalytically inactive but regulate the activity of other cell wall hydrolases and are classically described as cell separation factors NlpD and EnvC. Here, we explore the function of four LytM factors in the alphaproteobacterial plant pathogen Agrobacterium tumefaciens. An LmdC ortholog (Atu1832) and a MepM ortholog (Atu4178) are predicted to be catalytically active. While Atu1832 does not have an obvious function in cell growth or division, Atu4178 is essential for polar growth and likely functions as a space-making endopeptidase that cleaves amide bonds in the peptidoglycan cell wall during elongation. The remaining LytM factors are degenerate EnvC and NlpD orthologs. Absence of these proteins results in striking phenotypes indicative of misregulation of cell division and growth pole establishment. The deletion of an amidase, AmiC, closely phenocopies the deletion of envC suggesting that EnvC might regulate AmiC activity. The NlpD ortholog DipM is unprecedently essential for viability and depletion results in the misregulation of early stages of cell division, contrasting with the canonical view of DipM as a cell separation factor. Finally, we make the surprising observation that absence of AmiC relieves the toxicity induced by dipM overexpression. Together, these results suggest EnvC and DipM may function as regulatory hubs with multiple partners to promote proper cell division and establishment of polarity. [ABSTRACT FROM AUTHOR]

ROS are known to be accumulated in stigmas of different species and can possibly perform different functions important for plant reproduction. Here we tested the assumption that one of their functions is to control membrane potential and provoke synthesis of unique proteins in germinating pollen.We used spectrofluorometry and spectrophotometry to detect H2O2 in stigma exudate, quantitative fluorescent microscopy of pollen tubes and flow cytometry of pollen protoplasts to reveal effects on membrane potential, and a label‐free quantification approach to study pollen proteome changes after H2O2 treatment.We found that in both growing pollen tubes and pollen protoplasts exudate causes plasmalemma hyperpolarization similar to that provoked by H2O2. This effect is abolished by catalase treatment and the ROS quencher, MnTMPP. Inhibitory analysis indicates probable participation of Ca2+‐ and K+‐conducting channels in the observed hyperpolarization. For a deeper understanding of pollen response, we analysed proteome alterations in H2O2‐treated pollen grains. We found 50 unique proteins and 20 differently accumulated proteins that are mainly involved in cell metabolism, energetics, protein synthesis and folding.Observed hyperpolarization and proteome alterations agree well with previously reported stimulation of pollen germination by H2O2 and sensitivity of Ca2+‐ and K+‐conducting channels to this ROS. Thus, H2O2 is one of the active substances in tobacco stigma exudate that stimulates various physiological processes in germinating pollen. [ABSTRACT FROM AUTHOR]

Abstract Background Fungal fermentation is used to produce a diverse repertoire of enzymes, chemicals, and drugs for various industries. During submerged cultivation, filamentous fungi form a range of macromorphologies, including dispersed mycelia, clumped aggregates, or pellets, which have critical implications for rheological aspects during fermentation, gas/nutrient transfer, and, thus, product titres. An important component of strain engineering efforts is the ability to quantitatively assess fungal growth phenotypes, which will drive novel leads for morphologically optimized production strains. Results In this study, we developed an automated image analysis pipeline to quantify the morphology of pelleted and dispersed growth (MPD) which rapidly and reproducibly measures dispersed and pelleted macromorphologies from any submerged fungal culture. It (i) enables capture and analysis of several hundred images per user/day, (ii) is designed to quantitatively assess heterogeneous cultures consisting of dispersed and pelleted forms, (iii) gives a quantitative measurement of culture heterogeneity, (iv) automatically generates key Euclidian parameters for individual fungal structures including particle diameter, aspect ratio, area, and solidity, which are also assembled into a previously described dimensionless morphology number MN, (v) has an in-built quality control check which enables end-users to easily confirm the accuracy of the automated calls, and (vi) is easily adaptable to user-specified magnifications and macromorphological definitions. To concomitantly provide proof of principle for the utility of this image analysis pipeline, and provide new leads for morphologically optimized fungal strains, we generated a morphological mutant in the cell factory Aspergillus niger based on CRISPR-Cas technology. First, we interrogated a previously published co-expression networks for A. niger to identify a putative gamma-adaptin encoding gene (aplD) that was predicted to play a role in endosome cargo trafficking. Gene editing was used to generate a conditional aplD expression mutant under control of the titratable Tet-on system. Reduced aplD expression caused a hyperbranched growth phenotype and diverse defects in pellet formation with a putative increase in protein secretion. This possible protein hypersecretion phenotype could be correlated with increased dispersed mycelia, and both decreased pellet diameter and MN. Conclusion The MPD image analysis pipeline is a simple, rapid, and flexible approach to quantify diverse fungal morphologies. As an exemplar, we have demonstrated that the putative endosomal transport gene aplD plays a crucial role in A. niger filamentous growth and pellet formation during submerged culture. This suggests that endocytic components are underexplored targets for engineering fungal cell factories.

Polarized growth of pollen tubes is a critical step for successful reproduction in angiosperms and is controlled by ROP GTPases. Spatiotemporal activation of ROP (Rho GTPases of plants) necessitates a complex and sophisticated regulatory system, in which guanine nucleotide exchange factors (RopGEFs) are key components. It was previously shown that a leucine-rich repeat receptor-like kinase, Arabidopsis pollen receptor kinase 2 (AtPRK2), interacted with RopGEF12 for its membrane recruitment. However, the mechanisms underlying AtPRK2-mediated ROP activation in vivo are yet to be defined. It is reported here that over-expression of AtPRK2 induced tube bulging that was accompanied by the ectopic localization of ROP-GTP and the ectopic distribution of actin microfilaments. Tube depolarization was also induced by a potentially kinase-dead mutant, AtPRK2K366R, suggesting that the over-expression effect of AtPRK2 did not require its kinase activity. By contrast, deletions of non-catalytic domains in AtPRK2, i.e. the juxtamembrane (JM) and carboxy-terminal (CT) domains, abolished its ability to affect tube polarization. Notably, AtPRK2K366R retained the ability to interact with RopGEF12, whereas AtPRK2 truncations of these non-catalytic domains did not. Lastly, it has been shown that the JM and CT domains of AtPRK2 were not only critical for its interaction with RopGEF12 but also critical for its distribution at the plasma membrane. These results thus provide further insight into pollen receptor kinase-mediated ROP activation during pollen tube growth.

Plant peptides secreted as signal molecular to trigger cell-to-cell signaling are indispensable for plant growth and development. Successful sexual reproduction in plants requires extensive communication between male and female gametophytes, their gametes, and with the surrounding sporophytic tissues. In the past decade, it has been well-documented that small peptides participate in many important reproductive processes such as self-incompatibility, pollen tube growth, pollen tube guidance, and gamete interaction. Here, we provide a comprehensive overview of the peptides regulating the processes of male-female crosstalk in plant, aiming at systematizing the knowledge on the sexual reproduction, and signaling of plant peptides in future.

Plant peptides secreted as signal molecular to trigger cell-to-cell signaling are indispensable for plant growth and development. Successful sexual reproduction in plants requires extensive communication between male and female gametophytes, their gametes, and with the surrounding sporophytic tissues. In the past decade, it has been well-documented that small peptides participate in many important reproductive processes such as self-incompatibility, pollen tube growth, pollen tube guidance, and gamete interaction. Here, we provide a comprehensive overview of the peptides regulating the processes of male-female crosstalk in plant, aiming at systematizing the knowledge on the sexual reproduction, and signaling of plant peptides in future. [ABSTRACT FROM AUTHOR]

Endosome trafficking has been reported to play an essential role in pollen tube polar growth and NtGNL1 (Nicotiana tabacum GNOM-LIKE 1) regulates the polar growth through endosome trafficking. However, the regulation network and detailed molecular mechanisms underlying endosome trafficking remain unclear. Here, comparative proteomic analysis was carried out to survey the overall effect of NtGNL1 on pollen tube polar growth and NtGNL1-dependent endosome trafficking. With multiple comparative systems (RNAi, Wild type, and BFA or wortmannin treatments), 481 distinct proteins were identified including 43 common DEPs (differentially expressed proteins), of which 16 significant DEPs were common among RNAi, BFA, and wortmannin treated pollen tubes, indicating their close relation to the endosome trafficking. GO annotation indicates that the vesicle trafficking of gnl1 HE pollen tubes differs from that of the BFA and wortmannin treated pollen tubes in the COPII-coated vesicle budding process. KEGG pathway analysis suggests that the Pentose phosphate pathway is critical for the NtGNL1-dependent endosome trafficking. Yeast two-hybrid further confirmed that the NtGNL1-Sec7 domain interacted strongly with VPS32.2, TCTP, PIS2, and PDIL2-1, suggesting that the core functional region of NtGNL1 is the Sec7 domain. Therefore, NtGNL1 likely functions via its Sec7 binding with these proteins to affect endosome trafficking. Our results provide a clear outline of proteins involving in NtGNL1-dependent endosome trafficking and valuable clues for understanding the regulatory mechanism of NtGNL1 guided pollen tube polar growth. • Multiple comparative proteomic analysis was performed to survey the overall effect of NtGNL1 on pollen tube. • Sixteen DEPs were screened among RNAi, BFA and wortmannin treated pollen tubes. • GO indicates the RNAi pollen tubes differ from that of the chemical treated in the COPII-coated vesicle budding process. • NtGNL1 likely functions via its Sec7 binding to 4 proteins to affect endosome trafficking. [ABSTRACT FROM AUTHOR]

The genetic identities of Ca2+ channels in root hair (RH) tips essential for constitutive RH growth have remained elusive for decades. Here, we report the identification and characterization of three cyclic nucleotide-gated channel (CNGC) family members, CNGC5, CNGC6, and CNGC9, as Ca2+ channels essential for constitutive RH growth in Arabidopsis. We found that the cngc5-1cngc6-2cngc9-1 triple mutant (designated shrh1) showed significantly shorter and branching RH phenotypes as compared with the wild type. The defective RH growth phenotype of shrh1 could be rescued by either the expression of CNGC5, CNGC6, or CNGC9 single gene or by the supply of high external Ca2+, but could not be rescued by external K+ supply. Cytosolic Ca2+ imaging and patch-clamp data in HEK293T cells showed that these three CNGCs all function as Ca2+-permeable channels. Cytosolic Ca2+ imaging in growing RHs further showed that the Ca2+ gradients and their oscillation in RH tips were dramatically attenuated in shrh1 compared with those in the wild type. Phenotypic analysis revealed that these three CNGCs are Ca2+ channels essential for constitutive RH growth, with different roles in RHs from the conditional player CNGC14. Moreover, we found that these three CNGCs are involved in auxin signaling in RHs. Taken together, our study identified CNGC5, CNGC6, and CNGC9 as three key Ca2+ channels essential for constitutive RH growth and auxin signaling in Arabidopsis. Video Abstract:

The pH/T duality of acidic pH and temperature (T) action for the growth of grass shoots was examined in order to derive the phenomenological equation of wall properties for living plants. By considering non-meristematic growth as a dynamic series of state transitions (STs) in the extending primary wall, the critical exponents were identified, which exhibit a singular behaviour at a critical temperature, critical pH and critical chemical potential (μ) in the form of four power laws: f π τ ∝ τ β - 1 , f τ (π) ∝ π 1 - α , g μ (τ) ∝ τ - 2 - α + 2 β and g τ (μ) ∝ μ 2 - α . The indices α and β are constants, while π and τ represent a reduced pH and reduced temperature, respectively. The convexity relation α + β ≥ 2 for practical pH-based analysis and β ≡ 2 "mean-field" value in microscopic (μ) representation were derived. In this scenario, the magnitude that is decisive is the chemical potential of the H+ ions, which force subsequent STs and growth. Furthermore, observation that the growth rate is generally proportional to the product of the Euler beta functions of T and pH, allowed to determine the hidden content of the Lockhart constant Ф. It turned out that the pH-dependent time evolution equation explains either the monotonic growth or periodic extension that is usually observed—like the one detected in pollen tubes—in a unified account. [ABSTRACT FROM AUTHOR]

Owing to the local enrichment of factors that influence its dynamics and organization, the actin cytoskeleton displays different shapes and functions within the same cell. In yeast cells, post-Golgi vesicles ride on long actin cables to the bud tip. The proteins Boi1 and Boi2 (Boi1/2) participate in tethering and docking these vesicles to the plasma membrane. Here, we show in Saccharomyces cerevisiae that Boi1/2 also recruit nucleation and elongation factors to form actin filaments at sites of exocytosis. Disrupting the connection between Boi1/2 and the nucleation factor Bud6 impairs filament formation, reduces the directed movement of the vesicles to the tip and shortens the vesicles' tethering time at the cortex. Transplanting Boi1 from the bud tip to the peroxisomal membrane partially redirects the actin cytoskeleton and the vesicular flow towards the peroxisome, and creates an alternative, rudimentary vesicle-docking zone. We conclude that Boi1/2, through interactions with Bud6 and Bni1, induce the formation of a cortical actin structure that receives and aligns incoming vesicles before fusion with the membrane. [ABSTRACT FROM AUTHOR]

The accessibility to next-generation sequencing (NGS) techniques has enabled the sequencing of hundreds of genomes of species representing all kingdoms. In the case of fungi, genomes of more than a thousand of species are publicly available. This is far from covering the number of 2.2–3.8 million fungal species estimated to populate the world but has significantly improved the resolution of the fungal tree of life. Furthermore, it has boosted systematic evolutionary analyses, the development of faster and more accurate diagnostic analyses of pathogenic strains or the improvement of several biotechnological processes. Nevertheless, the diversification of the nature of fungal species used as model has also weakened research in other species that were traditionally used as reference in the pre-genomic era. In this context, and after more than 65 years since the first works published by Pontecorvo, Aspergillus nidulans remains as one of the most referential model filamentous fungus in research fields such as hyphal morphogenesis, intracellular transport, developmental programs, secondary metabolism, or stress response. This mini-review summarizes how A. nidulans has contributed to the progress in these fields during the last years, and discusses how it could contribute in the future, assisted by NGS and new-generation molecular, microscopy, or cellular tools. [ABSTRACT FROM AUTHOR]

The polarisome comprises a network of proteins that organizes polar growth in yeast and filamentous fungi. The yeast formin Bni1 and the actin nucleation-promoting factor Bud6 are subunits of the polarisome that together catalyze the formation of actin cables below the tip of yeast cells. We identified YFR016c (Aip5) as an interaction partner of Bud6 and the polarisome scaffold Spa2. Yeast cells lacking Aip5 display a reduced number of actin cables. Aip5 binds with its N-terminal region to Spa2 and with its C-terminal region to Bud6. Both interactions collaborate to localize Aip5 at bud tip and neck, and are required to stimulate the formation of actin cables. Our experiments characterize Aip5 as a novel subunit of a complex that regulates the number of actin filaments at sites of polar growth.

Importance: The genus of Mycobacterium includes important clinical pathogens ( M. tuberculosis ). Bacteria of this genus share the unusual features of their cell cycle such as asymmetric polar cell elongation and long generation time. Markedly, control of the mycobacterial cell cycle still remains not fully understood. The main cell growth determinant in mycobacteria is the essential protein DivIVA, which is also involved in cell division. DivIVA activity is controlled by phosphorylation, but the mechanism and significance of this process are unknown. Here, we show how the previously established protein interaction partner of DivIVA in mycobacteria, the segregation protein ParA, affects the DivIVA subcellular distribution. We also demonstrate the role of a newly identified M. smegmatis DivIVA and ParA interaction partner, a protein named PapM, and we establish how their interactions are modulated by phosphorylation. Demonstrating that the tripartite interplay affects the mycobacterial cell cycle contributes to the general understanding of mycobacterial growth regulation., Competing Interests: The authors declare no conflict of interest.

Summary: Calcium gradients underlie polarization in eukaryotic cells. In plants, a tip‐focused Ca2+‐gradient is fundamental for rapid and unidirectional cell expansion during epidermal root hair development. Here we report that three members of the cyclic nucleotide‐gated channel family are required to maintain cytosolic Ca2+ oscillations and the normal growth of root hairs. CNGC6, CNGC9 and CNGC14 were expressed in root hairs, with CNGC9 displaying the highest root hair specificity. In individual channel mutants, morphological defects including root hair swelling and branching, as well as bursting, were observed. The developmental phenotypes were amplified in the three cngc double mutant combinations. Finally, cngc6/9/14 triple mutants only developed bulging trichoblasts and could not form normal root hair protrusions because they burst after the transition to the rapid growth phase. Prior to developmental defects, single and double mutants showed increasingly disturbed patterns of Ca2+ oscillations. We conclude that CNGC6, CNGC9 and CNGC14 fulfill partially but not fully redundant functions in generating and maintaining tip‐focused Ca2+ oscillations, which are fundamental for proper root hair growth and polarity. Furthermore, the results suggest that these calmodulin‐binding and Ca2+‐permeable channels organize a robust tip‐focused oscillatory calcium gradient, which is not essential for root hair initiation but is required to control the integrity of the root hair after the transition to the rapid growth phase. Our findings also show that root hairs possess a large ability to compensate calcium‐signaling defects, and add new players to the regulatory network, which coordinates cell wall properties and cell expansion during polar root hair growth. Significance Statement: Although Ca2+ oscillations are required for tip growth in plants, the molecular mechanism underlying these signals has not been fully understood. We identified three members of the family of cyclic nucleotide‐gated cation channels, CNGC6, CNGC9 and CNGC14, that shape the Ca2+ oscillations in the root hair tip, and in turn prevent root hairs from bursting during rapid cell elongation. [ABSTRACT FROM AUTHOR]

Direct growth of a pollen tube is an effective mechanism of sperm delivery characteristic for the majority of seed plants. In most cases, only one tube grows from one grain to perform the delivery function; meanwhile in Picea the appearance of two tubes from a single pollen grain is quite common during in vitro germination. Here, we describe the phenomenon of bipolar germination and test two hypotheses on its nature and possible role in gametophyte functioning. The hypothesis on "trophic" function of multiple tubes provoked by poor nutrition discussed in literature was not confirmed by in vitro growth tests; bipolar germination strongly decreased with lowering sucrose availability. The highest proportion of bipolar germination occurred in optimal conditions. We then assumed that bipolar germination occurs because turgor pressure is a non-directional force and effective systems of cell wall mechanical regulation are lacking. In hypertonic medium, bipolar germination was sufficiently lower than in isotonic medium, which was consistent with prediction of the «mechanical» hypothesis. Scanning electron microscopy and fluorescence microscopy analysis of pollen morphology and cell wall dynamics during both types of germination showed that the appearance of a single tube or bipolar germination depends on the extension of exine rupture. Cell wall softening by short-term ·OH treatment sufficiently decreased the percent of bipolar germination without affecting total germination efficiency. We concluded that mechanical properties of the cell wall and turgor pressure could shift the balance towards one of the germination patterns. [ABSTRACT FROM AUTHOR]

In most well-studied rod-shaped bacteria, peptidoglycan is primarily crosslinked by penicillin-binding proteins (PBPs). However, in mycobacteria, crosslinks formed by L,D-transpeptidases (LDTs) are highly abundant. To elucidate the role of these unusual crosslinks, we characterized Mycobacterium smegmatis cells lacking all LDTs. We find that crosslinks generate by LDTs are required for rod shape maintenance specifically at sites of aging cell wall, a byproduct of polar elongation. Asymmetric polar growth leads to a non-uniform distribution of these two types of crosslinks in a single cell. Consequently, in the absence of LDT-mediated crosslinks, PBP-catalyzed crosslinks become more important. Because of this, Mycobacterium tuberculosis (Mtb) is more rapidly killed using a combination of drugs capable of PBP- and LDT- inhibition. Thus, knowledge about the spatial and genetic relationship between drug targets can be exploited to more effectively treat this pathogen.

ABSTRACT Agrobacterium tumefaciens grows by addition of peptidoglycan (PG) at one pole of the bacterium. During the cell cycle, the cell needs to maintain two different developmental programs, one at the growth pole and another at the inert old pole. Proteins involved in this process are not yet well characterized. To further characterize the role of pole-organizing protein A. tumefaciens PopZ (PopZAt), we created deletions of the five PopZAt domains and assayed their localization. In addition, we created a popZAt deletion strain (ΔpopZAt) that exhibited growth and cell division defects with ectopic growth poles and minicells, but the strain is unstable. To overcome the genetic instability, we created an inducible PopZAt strain by replacing the native ribosome binding site with a riboswitch. Cultivated in a medium without the inducer theophylline, the cells look like ΔpopZAt cells, with a branching and minicell phenotype. Adding theophylline restores the wild-type (WT) cell shape. Localization experiments in the depleted strain showed that the domain enriched in proline, aspartate, and glutamate likely functions in growth pole targeting. Helical domains H3 and H4 together also mediate polar localization, but only in the presence of the WT protein, suggesting that the H3 and H4 domains multimerize with WT PopZAt, to stabilize growth pole accumulation of PopZAt. IMPORTANCE Agrobacterium tumefaciens is a rod-shaped bacterium that grows by addition of PG at only one pole. The factors involved in maintaining cell asymmetry during the cell cycle with an inert old pole and a growing new pole are not well understood. Here we investigate the role of PopZAt, a homologue of Caulobacter crescentus PopZ (PopZCc), a protein essential in many aspects of pole identity in C. crescentus. We report that the loss of PopZAt leads to the appearance of branching cells, minicells, and overall growth defects. As many plant and animal pathogens also employ polar growth, understanding this process in A. tumefaciens may lead to the development of new strategies to prevent the proliferation of these pathogens. In addition, studies of A. tumefaciens will provide new insights into the evolution of the genetic networks that regulate bacterial polar growth and cell division.

Emerging evidence indicates that some reactive oxygen species (ROS), such as the superoxide anion radical and hydrogen peroxide (H2O2), are central regulators of plant responses to biotic and abiotic stresses. Thus, the cellular levels of ROS are thought to be tightly regulated by an efficient and elaborate pro- and antioxidant system that modulates the production and scavenging of ROS. Until recently, studies of ROS in plant cells have been limited to biochemical assays and the use of fluorescent probes; however, the irreversible oxidation of these fluorescent probes makes it impossible to visualize dynamic changes in ROS levels. In this work, we describe the use of Hyper, a recently developed live cell probe for H2O2 measurements in living cells, to monitor oxidative stress in Arabidopsis roots subjected to aluminum treatment. Hyper consists of a circularly permuted YFP (cpYFP) inserted into the regulatory domain of the Escherichia coli hydrogen peroxide-binding protein (OxyR), and is a H2O2-specific ratiometric, and therefore quantitative, probe that can be expressed in plant and animal cells. Now we demonstrate that H2O2 levels drop sharply in the elongation zone of roots treated with aluminum. This response could contribute to root growth arrest and provides evidence that H2O2 is involved in early Al sensing.

The mycobacterial cell envelope is a complex multilayered structure that provides the strength to the rod-shaped cell and creates the permeability barrier against antibiotics and host immune attack. In this review, we will discuss the spatial coordination of cell envelope biosynthesis and how plasma membrane compartmentalization plays a role in this process. The spatial organization of cell envelope biosynthetic enzymes as well as other membrane-associated proteins is crucial for cellular processes such as polar growth and midcell septum formation. We will highlight metabolic enzymes involved in the localized biosynthesis of envelope components such as peptidoglycan, arabinogalactan and outer/inner membrane lipids. The known and potential roles of cytoskeletal and coiled coil proteins in driving subcellular protein localization will also be summarized. Finally, we provide a comprehensive overview of known lateral heterogeneities in mycobacterial plasma membrane, with a particular focus on the intracellular membrane domain, recently revealed by biochemical fractionation and fluorescence microscopy. We consider how this dynamic and multifunctional membrane microdomain contributes to the subcellular localization of membrane proteins and spatially restricted cell envelope biosynthesis in mycobacteria. [ABSTRACT FROM AUTHOR]