The CHY-type zinc finger protein MoChy1 is involved in polarized growth, conidiation, autophagy and pathogenicity of Magnaporthe oryzae.

Polarized growth is critical for the development of filamentous phytopathogens, and the CHY-type zinc finger protein Chy1 regulates microtubule assembly to influence polarized growth and thereby affect plant infections. However, the biological role of a Chy1 homolog MoChy1 remains unknown in Magnaporthe oryzae. We found here that the MoChy1-GFP was distributed in the cytoplasm outside the vacuole in hyphae and localized mainly to the vacuole compartments as the appressorium matured. The Mochy1 mutants showed an extremely slow growth rate, curved and branched mycelium, reduced conidiation, and a smaller size in the appressorium. Meanwhile, the Mochy1 mutants showed increased sensitivity to benomyl, damaged microtubule cytoskeleton, and mislocalized polarisome protein MoSpa2 and chitin synthase MoChs6 in hyphae. Compared to Guy11, the Mochy1 mutants exhibited increased sensitivity to H2O2, impaired ability to eliminate host-derived ROS and reduced penetration into host plants, resulting in a strong reduction in pathogenicity of Mochy1 mutants. Furthermore, the Mochy1 mutants also exhibited defects in chitin distribution, osmotic stress tolerance, and septin ring organization during appressorium differentiation and fungal development. Nonselective autophagy was negatively regulated in Mochy1 mutants compared to Guy11. In summary, MoChy1 plays multiple roles in fungal polar growth and full virulence of M. oryzae.

Synergistic interplay of redox homeostasis and polysaccharide synthesis promotes cotton fiber elongation.

Cell polarity is the foundation of cell development and tissue morphogenesis. The investigation of polarized growth provides opportunities to gain profound insights into morphogenesis and tissue functionality in organisms. Currently, there are still many mysteries surrounding the mechanisms that regulate polarized cell growth. Cotton fiber cells serve as an excellent model for studying polarized growth, and provide important clues for unraveling the molecular mechanisms, signaling pathways, and regulatory networks of polarized growth. In this study, we characterized two functional genes, GhMDHAR1AT/DT and GhDHAR2AT/DT with predominant expression during fiber elongation. Loss of function of both genes contributed to a significant increase in fiber length. Transcriptomic data revealed up-regulated expression of antioxidant genes in CRISPR mutant lines, along with delayed expression of secondary wall-related genes and temporally prolonged expression of primary wall-related genes. Experimental evidence demonstrated that the increase in GSH content and glutathione peroxidase (GPX) enzyme activity led to enhanced total antioxidant capacity (T-AOC), resulting in reduced H2O2 levels, which contributed to the extension of fiber elongation stage in CRISPR mutant lines. Moreover, the increased polysaccharide synthesis in CRISPR mutant lines was found to provide an abundant supply of raw materials for fiber cell wall elongation, suggesting that synergistic interplay between redox homeostasis and polysaccharide synthesis in fiber cells may facilitate cell wall remodeling and fiber elongation. This study provides valuable insights for deciphering the mechanisms of cell polarized growth and improving cotton fiber quality.

The small Ras-like GTPase BUD-1 modulates conidial germination and hyphal growth guidance in the filamentous fungus Neurospora crassa.

In filamentous fungi, the hypha orientation is essential for polarized growth and morphogenesis. The ability to re-orient tip growth in response to environmental cues is critical for the colony survival. Therefore, hyphal tip orientation and tip extension are distinct mechanisms that operate in parallel during filamentous growth. In yeast, the axial growth orientation requires a pathway regulated by Rsr1p/Bud1p, a Ras-like GTPase protein, which determines the axial budding pattern. However, in filamentous fungi the function of the Rsr1/Bud1p gene (krev-1 homolog) has not been completely characterized. In this work, we characterized the phenotype of a homokaryon mutant Bud1p orthologous in Neurospora crassa (△bud-1) and tagged BUD-1 with the green fluorescent protein (GFP) to determine its localization and cell dynamics under confocal microscopy. During spore germination BUD-1 was localized at specific points along the plasma membrane and during germ tube emergence it was located at the tip of the germ tubes. In mature hyphae BUD-1 continued to be located at the cell tip and was also present at sites of branch emergence and at the time of septum formation. The △bud-1 mutant showed a delayed germination, and the orientation of hyphae was somewhat disrupted. Also, the hypha diameter was reduced approximately 37 % with respect to the wild type. The lack of BUD-1 affected the Spitzenkörper (Spk) formation, trajectory, the localization of polarisome components BNI-1 and SPA-2, and the actin cytoskeleton polarization. The results presented here suggest that BUD-1 participates in the establishment of a new polarity axis. It may also mediate the delivery of secretory vesicles for the efficient construction of new plasma membrane and cell wall.

Stage-Specific Genetic Interaction between FgYCK1 and FgBNI4 during Vegetative Growth and Conidiation in Fusarium graminearum.

CK1 casein kinases are well conserved in filamentous fungi. However, their functions are not well characterized in plant pathogens. In Fusarium graminearum, deletion of FgYCK1 caused severe growth defects and loss of conidiation, fertility, and pathogenicity. Interestingly, the Fgyck1 mutant was not stable and often produced fast-growing spontaneous suppressors. Suppressor mutations were frequently identified in the FgBNI4 gene by sequencing analyses. Deletion of the entire FgBNI4 or disruptions of its conserved C-terminal region could suppress the defects of Fgyck1 in hyphal growth and conidiation, indicating the genetic relationship between FgYCK1 and FgBNI4. Furthermore, the Fgyck1 mutant showed defects in polarized growth, cell wall integrity, internalization of FgRho1 and vacuole fusion, which were all partially suppressed by deletion of FgBNI4. Overall, our results indicate a stage-specific functional relationship between FgYCK1 and FgBNI4, possibly via FgRho1 signaling for regulating polarized hyphal growth and cell wall integrity.

NADPH Oxidases Play a Role in Pathogenicity via the Regulation of F-Actin Organization in Colletotrichum gloeosporioides.

Multiunit-flavoenzyme NADPH oxidases (NOXs) play multiple roles in living cells via regulating signaling pathways. In several phytopathogenic fungi, NOXs are required for the polarized growth of hyphal tips and pathogenicity to host plants, but the possible mechanisms are still elusive. In our previous study, CgNOXA, CgNOXB, and CgNOXR were identified as components of the NOX complex in Colletotrichum gloeosporioides. The growth and the inoculation assays revealed that CgNOXA/B and CgNOXR regulate vegetative growth and are required for the full pathogenicity of C. gloeosporioides to Hevea leaves. We further demonstrated that the vital roles of CgNOXB and CgNOXR in appressorium formation and the development of invasion hyphae account for their functions in pathogenicity. Moreover, CgNOXB and CgNOXR regulate the production and distribution of ROS in hyphal tips and appressoria, control the specialized remodeling of F-actin in hyphal tips and appressoria, and are involved in fungal cell wall biosynthesis. Taken together, our findings highlight the role of NOXs in fungal pathogenicity through the organization of the actin cytoskeleton.

The Fungal Protein Mes1 Is Required for Morphogenesis and Virulence in the Dimorphic Phytopathogen Ustilago maydis.

Polarized growth is a defining property of filamentous fungi, which plays an important role in different aspects of their biology, including virulence. However, little information is available about the determinants of cell surface organization and their role in polarized growth. The fungal protein MesA was identified in a genetic screen in Aspergillus nidulans and is involved in the stabilization of the polarity axes, but it has no evident role in budding yeast. In this work, I present evidence that in the dimorphic fungal phytopathogen Ustilago maydis MesA/Mes1 is involved in cell wall stability and polarized growth. mes1 mutants were more sensitive to drugs provoking cell wall stress, and they displayed a temperature-sensitive phenotype. Actin cytoskeleton was disorganized in a mes1 mutant, suggesting that there is a connection between Mes1, the actin cytoskeleton and polarized morphogenesis. The septin ring was also absent from the bud tip, but not the bud neck. Deletion of mes1 provoked defects in endocytosis and vacuolar organization in the cells. Mes1 was essential for strong polarized growth in the hyphal form, but it was dispensable during low or moderate polarized growth in the yeast form in U. maydis at a permissive temperature. Consistently, mes1 mutants showed delayed mating and they were avirulent.

F-actin dynamics following mechanical injury of Trichoderma atroviride and Neurospora crassa hyphae.

We investigated hyphae regeneration in Trichoderma atroviride and Neurospora crassa, with particular focus on determining the role of the actin cytoskeleton after mechanical injury. Filamentous actin (F-actin) dynamics was observed by live-cell confocal microscopy in both T. atroviride and N. crassa strains expressing Lifeact-GFP. In growing hyphae of both fungi, F-actin localized in three different structural forms: patches, cables and actomyosin rings. Most patches were conspicuously arranged in a collar in the hyphal subapex. A strong F-actin signal, likely actin filaments, colocalized with the core of the Spitzenkörper. Filaments and cables of F-actin were observed along the cortex throughout hyphae. Following mechanical damage at the margin of growing mycelia of T. atroviride and N. crassa, the severed hyphae lost their cytoplasmic contents, but plugging of the septal pore by a Woronin body occured, and the rest of the hyphal tube remained whole. In both fungi, patches of F-actin began accumulating next to the plugged septum. Regeneration was attained by the emergence of a new hyphal tube as an extension of the plugged septum wall. The septum wall was gradually remodeled into the apical wall of the emerging hypha. Whereas in T. atroviride the re-initiation of polarized growth took  âˆ¼ 1 h, in N. crassa, actin patch accumulation began almost immediately, and new growing hyphae were observed âˆ¼ 30 min after injury. By confocal microscopy, we found that chitin synthase 1 (CHS-1), a microvesicle (chitosome) component, accumulated next to the plugged septum in regenerating hyphae of N. crassa. We concluded that the actin cytoskeleton plays a key role in hyphal regeneration by supporting membrane remodeling, helping to facilitate transport of vesicles responsible for new wall growth and organization of the new tip-growth apparatus.

Expanding the Biological Role of Lipo-Chitooligosaccharides and Chitooligosaccharides in Laccaria bicolor Growth and Development.

The role of lipo-chitooligosaccharides (LCOs) as signaling molecules that mediate the establishment of symbiotic relationships between fungi and plants is being redefined. New evidence suggests that the production of these molecular signals may be more of a common trait in fungi than what was previously thought. LCOs affect different aspects of growth and development in fungi. For the ectomycorrhizal forming fungi, Laccaria bicolor, the production and effects of LCOs have always been studied with a symbiotic plant partner; however, there is still no scientific evidence describing the effects that these molecules have on this organism. Here, we explored the physiological, molecular, and metabolomic changes in L. bicolor when grown in the presence of exogenous sulfated and non-sulfated LCOs, as well as the chitooligomers, chitotetraose (CO4), and chitooctaose (CO8). Physiological data from 21 days post-induction showed reduced fungal growth in response to CO and LCO treatments compared to solvent controls. The underlying molecular changes were interrogated by proteomics, which revealed substantial alterations to biological processes related to growth and development. Moreover, metabolite data showed that LCOs and COs caused a downregulation of organic acids, sugars, and fatty acids. At the same time, exposure to LCOs resulted in the overproduction of lactic acid in L. bicolor. Altogether, these results suggest that these signals might be fungistatic compounds and contribute to current research efforts investigating the emerging impacts of these molecules on fungal growth and development.

ROPs: Molecular Switches of Multiple Signal Pathways in Plant Cells / 中国生物化学与分子生物学报

RHO-related GTPases of plants (ROPs) are a class of signal transduction G proteins (alsoknown as GTP binding proteins) widely existing in plants. ROP proteins act as " molecular switches" toregulate the signal transduction process during cellular activities such as plant cell polarity regulation, plant morphological development, hormone level regulation, stress responses and many other life activitiesby shifting between inactive GDP-binding and active GTP-binding forms in the cells. In this review, thedomain structure, classification, the mechanism of activity regulation and biological functions of ROPproteins were summarize. Furthermore, ROP proteins from Arabidopsis, maize, rice and barley werephylogenetically analyzed. The results show that ROP proteins were classified into two types based on thedomain structure of the proteins. However, these ROP proteins were divided into 4 clades based on thesimilarity of protein sequences. Furthermore, the mechanism of ROP proteins as a molecular switchregulating various signaling pathways in cells, and the specific functions and mechanisms of ROPs in thepolarized growth of pollen tubes, root hairs and plant pavement cells and other stress responses werecharacterized. In addition, the research progress of the function of ROPs in plant hormones such as ABA, IAA and BR mediated signal transduction were described as well. At last, the unanswered questions suchas why different ROP proteins play distinct roles in the same signaling pathway and how ROPs coordinatedifferent signal pathways to jointly regulate a plant’ s development or physiological process werediscussed, which may shed light on future research.

[Formins: the key regulators of plant cell morphology and development].

Formins are widely distributed in eukaryotes such as fungi, plants and animals. They play crucial roles in regulating the polymerization of actin, coordinating the synergistic interactions between actin and microtubules, and determining cell growth and morphology. Unlike formins from fungi and animals, plant formins have been evolved into two plant-specific types. Generally, type Ⅱ formins are believed to regulate the polarized growth of cells, and type Ⅰ formins may regulate the cell expansion and division processes. Recent studies on the function of plant formins suggest it is inappropriate to classify the function of formins purely based on their structures. This review summarizes the domain organization of formins and their corresponding functions, as well as the underpinning mechanisms. Furthermore, the unsolved or unexplored issues along with future perspectives on plant formins are proposed and discussed.

Identifying Conserved Generic Aspergillus spp. Co-Expressed Gene Modules Associated with Germination Using Cross-Platform and Cross-Species Transcriptomics.

Aspergillus spp. is an opportunistic human pathogen that may cause a spectrum of pulmonary diseases. In order to establish infection, inhaled conidia must germinate, whereby they break dormancy, start to swell, and initiate a highly polarized growth process. To identify critical biological processes during germination, we performed a cross-platform, cross-species comparative analysis of germinating A. fumigatus and A. niger conidia using transcriptional data from published RNA-Seq and Affymetrix studies. A consensus co-expression network analysis identified four gene modules associated with stages of germination. These modules showed numerous shared biological processes between A. niger and A. fumigatus during conidial germination. Specifically, the turquoise module was enriched with secondary metabolism, the black module was highly enriched with protein synthesis, the darkgreen module was enriched with protein fate, and the blue module was highly enriched with polarized growth. More specifically, enriched functional categories identified in the blue module were vesicle formation, vesicular transport, tubulin dependent transport, actin-dependent transport, exocytosis, and endocytosis. Genes important for these biological processes showed similar expression patterns in A. fumigatus and A. niger, therefore, they could be potential antifungal targets. Through cross-platform, cross-species comparative analysis, we were able to identify biologically meaningful modules shared by A. fumigatus and A. niger, which underscores the potential of this approach.

Quantitative cell biology of tip growth in moss.

KEY MESSAGE: Here we review, from a quantitative point of view, the cell biology of protonemal tip growth in the model moss Physcomitrium patens. We focus on the role of the cytoskeleton, vesicle trafficking, and cell wall mechanics, including reviewing some of the existing mathematical models of tip growth. We provide a primer for existing cell biological tools that can be applied to the future study of tip growth in moss. Polarized cell growth is a ubiquitous process throughout the plant kingdom in which the cell elongates in a self-similar manner. This process is important for nutrient uptake by root hairs, fertilization by pollen, and gametophyte development by the protonemata of bryophytes and ferns. In this review, we will focus on the tip growth of moss cells, emphasizing the role of cytoskeletal organization, cytoplasmic zonation, vesicle trafficking, cell wall composition, and dynamics. We compare some of the existing knowledge on tip growth in protonemata against what is known in pollen tubes and root hairs, which are better-studied tip growing cells. To fully understand how plant cells grow requires that we deepen our knowledge in a variety of forms of plant cell growth. We focus this review on the model plant Physcomitrium patens, which uses tip growth as the dominant form of growth at its protonemal stage. Because mosses and vascular plants shared a common ancestor more than 450 million years ago, we anticipate that both similarities and differences between tip growing plant cells will provide mechanistic information of tip growth as well as of plant cell growth in general. Towards this mechanistic understanding, we will also review some of the existing mathematical models of plant tip growth and their applicability to investigate protonemal morphogenesis. We attempt to integrate the conclusions and data across cell biology and physical modeling to our current state of knowledge of polarized cell growth in P. patens and highlight future directions in the field.

Lipid flippases in polarized growth.

Polarized growth is required in eukaryotic cells for processes such as cell division, morphogenesis and motility, which involve conserved and interconnected signalling pathways controlling cell cycle progression, cytoskeleton reorganization and secretory pathway functioning. While many of the factors involved in polarized growth are known, it is not yet clear how they are coordinated both spatially and temporally. Several lines of evidence point to the important role of lipid flippases in polarized growth events. Lipid flippases, which mainly belong to the P4 subfamily of P-type ATPases, are active transporters that move different lipids to the cytosolic side of biological membranes at the expense of ATP. The involvement of the Saccharomyces cerevisiae plasma membrane P4 ATPases Dnf1p and Dnf2p in polarized growth and their activation by kinase phosphorylation were established some years ago. However, these two proteins do not seem to be responsible for the phosphatidylserine internalization required for early recruitment of proteins to the plasma membrane during yeast mating and budding. In a recent publication, we demonstrated that the Golgi-localized P4 ATPase Dnf3p has a preference for PS as a substrate, can reach the plasma membrane in a cell cycle-dependent manner, and is regulated by the same kinases that activate Dnf1p and Dnf2p. This finding solves a long-lasting enigma in the field of lipid flippases and suggests that tight and heavily coordinated spatiotemporal control of lipid translocation at the plasma membrane is important for proper polarized growth.

FgSpa2 recruits FgMsb3, a Rab8 GAP, to the polarisome to regulate polarized trafficking, growth and pathogenicity in Fusarium graminearum.

In filamentous fungi, hyphal growth depends on the continuous delivery of vesicles to the growing tips. It is unclear how fast-growing hyphae coordinate simultaneous cell extension and expansion in the tip cells. We have functionally characterized 12 TBC (Tre-2/Bub2/Cdc16) domain-containing proteins in Fusarium graminearum. Among them, FgMsb3 is found to regulate hyphal tip expansion and to be required for pathogenicity. The regulatory mechanism of FgMsb3 has been further investigated by genetic, high-resolution microscopy and high-throughput co-immunoprecipitation strategies. The FgMsb3 protein localizes at the polarisome and the hyphal apical dome (HAD) where it acts as a GTPase-activating protein for FgRab8 which is required for apical secretion-mediated growth and pathogenicity. Deletion of FgMSB3 causes excessive polarized trafficking but blocks the fusion of FgSnc1-associated vesicles to the plasma membrane. Moreover, we establish that FgSpa2 interacts with FgMsb3, enabling FgMsb3 tethering to the polarisome. Loss of FgSpa2 or other polarisome components (FgBud6 and FgPea2) causes complete shifting of FgMsb3 to the HAD and this affects the polarized growth and pathogenicity of the fungus. In summary, we conclude that FgSpa2 regulates FgMsb3-FgRab8 cascade and this is crucial for creating a steady-state equilibrium that maintains continuous polarized growth and contributes to the pathogenicity of F. graminearum.

Formins: the key regulators of plant cell morphology and development / 生物工程学报

Formins are widely distributed in eukaryotes such as fungi, plants and animals. They play crucial roles in regulating the polymerization of actin, coordinating the synergistic interactions between actin and microtubules, and determining cell growth and morphology. Unlike formins from fungi and animals, plant formins have been evolved into two plant-specific types. Generally, type Ⅱ formins are believed to regulate the polarized growth of cells, and type Ⅰ formins may regulate the cell expansion and division processes. Recent studies on the function of plant formins suggest it is inappropriate to classify the function of formins purely based on their structures. This review summarizes the domain organization of formins and their corresponding functions, as well as the underpinning mechanisms. Furthermore, the unsolved or unexplored issues along with future perspectives on plant formins are proposed and discussed.

Galactomannan Produced by Aspergillus fumigatus: An Update on the Structure, Biosynthesis and Biological Functions of an Emblematic Fungal Biomarker.

The galactomannan (GM) that is produced by the human fungal pathogen Aspergillus fumigatus is an emblematic biomarker in medical mycology. The GM is composed of two monosaccharides: mannose and galactofuranose. The furanic configuration of galactose residues, absent in mammals, is responsible for the antigenicity of the GM and has favoured the development of ELISA tests to diagnose aspergillosis in immunocompromised patients. The GM that is produced by A. fumigatus is a unique fungal polysaccharide containing a tetramannoside repeat unit and having three different forms: (i) membrane bound through a glycosylphosphatidylinositol (GPI)-anchor, (ii) covalently linked to ß-1,3-glucans in the cell wall, or (iii) released in the culture medium as a free polymer. Recent studies have revealed the crucial role of the GM during vegetative and polarized fungal growth. This review highlights these recent data on its biosynthetic pathway and its biological functions during the saprophytic and pathogenic life of this opportunistic human fungal pathogen.

The Sur7/PalI family transmembrane protein Tos7 (Yol019w) plays a role in secretion in budding yeast.

Tos7 (Yol019w) is a Sur7/PalI family transmembrane protein in the budding yeast Saccharomyces cerevisiae. Since the deletion of TOS7 did not affect growth or cell morphology, the cellular roles of Tos7 have not been established previously. Here, we show that high-copy TOS7 expression suppressed the growth defect of the secretion-defective RGA1-C term-overexpressing mutant and sec15-1 mutant. Moreover, Tos7 physically interacted with Boi2 and the Rho GTPase Rho3, two key regulators of exocyst assembly, suggesting that Tos7 plays a role in secretion. We also show that the deletion of TOS7 rendered the cells more sensitive to the cell wall-disrupting agents Congo red and calcofluor white while high-copy TOS7 expression had an opposite effect, suggesting that Tos7 affects cell wall organization. Finally, we show that Tos7 localized to punctate patches on the plasma membrane that were largely co-localized with the plasma membrane microdomains named MCC (membrane compartment of Can1). Together, these results suggest that Tos7 contributes to cell surface-related functions. Tos7 is likely an auxiliary component of MCC/eisosome that specifically interacts with the secretory pathway.

GPCR receptor phosphorylation and endocytosis are not necessary to switch polarized growth between internal cues during pheromone response in S. cerevisiae.

Chemotactic/chemotropic cells follow accurately the direction of gradients of regulatory molecules. Many G-protein-coupled receptors (GPCR) function as chemoattractant receptors to guide polarized responses. In "a" mating type yeast, the GPCR Ste2 senses the α-cell's pheromone. Previously, phosphorylation and trafficking of this receptor have been implicated in the process of gradient sensing, where cells dynamically correct growth. Correction is often necessary since yeast have intrinsic polarity sites that interfere with a correct initial gradient decoding. We have recently showed that when actively dividing (not in G1) yeast are exposed to a uniform pheromone concentration, they initiate a pheromone-induced polarization next to the mother-daughter cytokinesis site. Then, they reorient their growth to the intrinsic polarity site. Here, to study if Ste2 phosphorylation and internalization are involved in this process, we generated receptor variants combining three types of mutated signals for the first time: phosphorylation, ubiquitylation and the NPFX1,2D Sla1-binding motif. We first characterized their effect on endocytosis and found that these processes regulate internalization in a more complex manner than previously shown. Interestingly, we showed that receptor phosphorylation can drive internalization independently of ubiquitylation and the NPFX1,2D motif. When tested in our assays, cells expressing either phosphorylation or endocytosis-deficient receptors were able to switch away from the cytokinesis site to find the intrinsic polarity site as efficiently as their WT counterparts. Thus, we conclude that these processes are not necessary for the reorientation of polarization.

Roles of the PH, coiled-coil and SAM domains of the yeast polarity protein Boi2 in polarity-site localization and function in polarized growth.

Boi1 and Boi2 are paralogous proteins essential for bud formation in budding yeast. So far, the domains that target Boi1/Boi2 to the polarity sites and function in bud formation are not well understood. Here, we report that a coiled-coil domain of Boi2 cooperates with the adjacent PH domain to confer Boi2's bud-cortex localization and major function in cell growth. The PH domain portion of the PH-CC bi-domain interacts with the Rho GTPases Cdc42 and Rho3 and both interactions are independent of the GTP/GDP-bound state of each GTPase. Interestingly, high-copy RHO3 and BOI2 but not CDC42 suppressed the growth defect of RGA1-C538 overexpression and the sec15-1 mutant and this BOI2 function depends on RHO3, suggesting that Boi2 may function in the Rho3 pathway. The SAM domain of Boi2 plays an essential role in high-copy suppression of the two mutants as well as in the early bud-neck localization of Boi2. The SAM domain and the CC domain also interact homotypically. They are likely involved in the formation of Boi2-containing protein complex. Our results provide new insights in the localization and function of Boi2 and highlight the importance of the PH-CC bi-domain and the SAM domain in Boi2's localization and function.