cells Review The Small GTPases in Fungal Signaling Conservation and Function Mitzuko Dautt-Castro * , Montserrat Rosendo-Vargas and Sergio Casas-Flores * Laboratorio de Genómica Funcional y Comparativa, División de Biología Molecular, IPICYT, San Luis Potosí 78216, Mexico; [email protected] * Correspondence: [email protected] (M.D.-C.); [email protected] (S.C.-F.) Abstract: Monomeric GTPases, which belong to the Ras superfamily, are small proteins involved in many biological processes. They are fine-tuned regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Several families have been identified in organisms from different kingdoms. Overall, the most studied families are Ras, Rho, Rab, Ran, Arf, and Miro. Recently, a new family named Big Ras GTPases was reported. As a general rule, the proteins of all families have five characteristic motifs (G1–G5), and some specific features for each family have been described. Here, we present an exhaustive analysis of these small GTPase families in fungi, using 56 different genomes belonging to different phyla. For this purpose, we used distinct approaches such as phylogenetics and sequences analysis. The main functions described for monomeric GTPases in fungi include morphogenesis, secondary metabolism, vesicle trafficking, and virulence, which are discussed here. Their participation during fungus–plant interactions is reviewed as well. Keywords: small GTPases; Ras; Rho; Rab; Ran; Arf; Miro; BRG; fungi Citation: Dautt-Castro, M.; Rosendo-Vargas, M.; Casas-Flores, S. 1. Overview The Small GTPases in Fungal Small Ras GTPases (rat sarcoma guanine triphosphatases) are monomeric G proteins Signaling Conservation and Function. of low molecular weight (21 to 30 kDa) that are present in all eukaryotes and that participate Cells 2021, 10, 1039. https://doi.org/ in many biological processes. They act as molecular switches, alternating between an active 10.3390/cells10051039 GTP-bound state and an inactive GDP-bound state [1]. This regulation is carried out through the activity of guanine-nucleotide exchange factors (GEFs) and GTPase activating Academic Editor: Bor Luen Tang proteins (GAPs) that catalyze the replacement of GDP by GTP and promote the inactive Received: 15 March 2021 GDP-bound form, respectively (Figure1)[2–4]. Accepted: 23 April 2021 These proteins have a conserved GTP/GDP core and a hydrolysis domain bearing Published: 28 April 2021 five characteristic and highly conserved motifs: G1 (Walker A/P-loop; GxxxxG K[S/T]) implicated in the binding of β- and γ-phosphate groups of the nucleotide; G2 (Switch I; 2+ Publisher’s Note: MDPI stays neutral x[T/S]x) that binds Mg , essential for GTP hydrolysis; G3 (Walker B/Switch II; DxxG) 2 with regard to jurisdictional claims in that interacts with the nucleotide γ-phosphate and Mg ; G4 ([N/T]KxD) that binds directly published maps and institutional affil- to the nucleotide at K and D sites; and G5 (S/CAK/L/T), involved in guanine base iations. recognition [5,6]. The switch I and II regions are flexible segments that sense the nucleotide state and allow the small GTPases to interact with downstream effectors [7,8]. Members of the Small Ras-GTPases superfamily take part in cell regulation, growth, morphogenesis, cell division, and virulence and are known as master regulators of intracel- Copyright: © 2021 by the authors. lular bidirectional vesicle transport [1,9]. They are classified according to their amino acid Licensee MDPI, Basel, Switzerland. sequence and their biochemical properties. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Cells 2021, 10, 1039. https://doi.org/10.3390/cells10051039 https://www.mdpi.com/journal/cells Cells 2021, 10, x FOR PEER REVIEW 2 of 29 Cells 2021, 10, 1039 2 of 29 Figure 1. Small GTPases ofof thethe RasRas superfamilysuperfamily regulate regulate several several processes processes in in fungi. fungi. In In fungal fungal cells, cells, GEFs GEFs and and GAPs GAPs catalyze cata- lyzealternation alternation between between an active an active GTP-bound GTP-bound state state and and an inactive an inactive GDP-bound GDP-bound state. state. Active Active GTPase GTPase interacts interacts with with specific spe- cificdownstream downstream effector, effector leading, leading to different to different cell responses. cell responses. The most The representative most representative functions functions of Ras GTPase of Ras familiesGTPase arefamilies listed arein the listed box. in the box. MembersAs shown of in the Figure Small1, Ras the- mostGTPases common superfamily families take and part their in main cell regulation, functions ingrowth, fungi morphogenesare Ras (rat sarcoma),is, cell division, associated and with virulence morphogenesis and are known and virulence as master of fungiregulators with differentof intra- cellularlifestyles, bidirectional such as pathogens, vesicle transport saprobes, [1,9] entomopathogens,. They are classified and mutualists; according Rho to their (Ras amino homo- acidlogue) sequence also known and their as Cdc42/Rho-GTPases, biochemical properties. involved in the regulation of cell polarity, gene expression,As shown and in membrane Figure 1, traffic;the most Rab common (Ras homologue families fromand their brain) main that functions participates in infungi the aresecretion Ras (rat of metabolitessarcoma), associated and lytic enzymeswith morphogenesis [10]; Arf (ADP and ribosylation virulence factors)of fungi participate with differ- in entmembrane/protein lifestyles, such as trafficking pathogens, to thesaprobes, plasma entomopathogens, membrane [9]; Ran and (Ras-like mutualists; nuclear Rho proteins) (Ras homologue)play pivotal rolesalso known in nucleocytoplasmic as Cdc42/Rho- andGTPas nucleares, involved pore transport, in the regulation synthesis of of cell RNA, polar- and ity,as a gene checkpoint expression, of the and cell membrane cycle [11]; traffic; and Miro Rab (mitochondrial (Ras homologue Rho-GTPases), from brain) that also partici- known patesas atypical in the Rhosecretion GTPases, of metabolites serves critical and lytic roles enzymes in mitochondria [10]; Arf (ADP morphology, ribosylation inheritance, factors) participateand homeostasis in membrane/protein [12]. Other less trafficking studied families to the haveplasma been membrane reported, [9] such; Ran as (Ras Era-like-like nuclear(Escherichia proteins) coli Ras-like play pivotal protein), roles involved in nucleocytoplasmic in ribosomal and assembly nuclear [13 pore], and transport, Spg1/Tem1 syn- thesis(Septum of RNA, promoting) and as that a checkpoint participate of in the ring cell constriction, cycle [11]; and septation, Miro (mitochondrial and cell division Rho in- GTPases)Schizosaccharomyces, also known pombe as at[14ypical]. Recently, Rho GTPases, a new fungal serves family critical of roles Ras GTPasesin mitochondria was reported, mor- phology,the Big Ras inheritance GTPases, (BRG), and homeostasis whose main [12] difference. Other isless their studied size, 3- families to 4-times have bigger been com- re- portedpared with, such the as classicalEra-like small(Escherichia GTPases coli as Ras well-like as protein) with their, involved poorly conserved in ribosomal G4 motif. assembly The [13]only, and member Spg1/Tem characterized1 (Septum until promoting) now, TBRG-1 that ofparticipate the plant mutualisticin ring constriction, fungus Trichoderma septation, andvirens cell, is division involved in in Schizosaccharomyces conidiation, development, pombe [14] secondary. Recently, metabolism, a new fungal mycoparasitism, family of Ras GTPasesand biocontrol was reported, [15]. Proteins the Big from Ras the GTPases different (BRG), families whose crosstalk main difference with each otheris their and size, with 3- members of other signal transduction pathways, which together comprise a very large and to 4-times bigger compared with the classical small GTPases as well as with their poorly complex network [9]. conserved G4 motif. The only member characterized until now, TBRG-1 of the plant mu- In this review, we present a comprehensive work where we discuss the state of the tualistic fungus Trichoderma virens, is involved in conidiation, development, secondary art regarding the main families of monomeric GTPases in fungi. Furthermore, we present metabolism, mycoparasitism, and biocontrol [15]. Proteins from the different families analyses at the genomic, phylogenetic, and sequence levels as well as the function of each crosstalk with each other and with members of other signal transduction pathways, which protein family. together comprise a very large and complex network [9]. 2. ClassificationIn this review, and we Phylogenetic present a comprehensive Conservation work of Small where Ras we GTPases discuss inthe Fungi state of the art regarding the main families of monomeric GTPases in fungi. Furthermore, we present Fungi are eukaryotic organisms that are highly relevant ecologically. They differ from analyses at the genomic, phylogenetic, and sequence levels as well as the function of each plants due to their heterotrophic nature, and from animals because they possess cell walls. protein family. Their closest kingdom is the Animalia, from which they diverged nearly 1.3 billion years ago [16,17]. One of their major features is osmotrophy, a way of feeding and nourishment Cells 2021, 10, 1039 3 of 29 that involves the displacement