Physiological Basis of Smut Infectivity in the Early Stages of Sugar Cane Colonization

Journal of Fungi

Review Physiological Basis of Smut Infectivity in the Early Stages of Sugar Cane Colonization

Carlos Vicente, María-Estrella Legaz * and Elena Sánchez-Elordi

Team of Intercellular Communication in Plant Symbiosis, Faculty of Biology, Complutense University, 12 José Antonio Novais Av., 28040 Madrid, Spain; [email protected] (C.V.); [email protected] (E.S.-E.) * Correspondence: [email protected]; Tel.: +34-1-3944565

Abstract: Sugar cane smut (Sporisorium scitamineum) interactions have been traditionally considered from the plant’s point of view: How can resistant sugar cane plants defend themselves against smut disease? Resistant plants induce several defensive mechanisms that oppose fungal attacks. Herein, an overall view of Sporisorium scitamineum’s mechanisms of infection and the defense mechanisms of plants are presented. Quorum sensing effects and a continuous reorganization of cytoskeletal components, where actin, myosin, and microtubules are required to work together, seem to be some of the keys to a successful attack.

Keywords: actin; cytoskeleton; infectivity; myosin; quorum sensing; smut; tubulin

1. Introduction Sugarcane plants are affected by multiple microorganisms, where among them is Sporisorium scitamineum (Syd.) M. Piepenbr., M. Stoll, and F. Oberw. (Ustilago scitaminea

Sydow), the causal agent of smut. It currently occurs in over 64 countries and sugarcane regions, in many of which, it causes a signiﬁcant amount of damage [1]. This damage

Citation: Vicente, C.; Legaz, M.-E.; increases in the sick sprouts due to secondary infections, and an increase in the size of the Sánchez-Elordi, E. Physiological Basis inoculum occurs when the whips break (a mixture of plant and fungal tissues, which is of Smut Infectivity in the Early Stages a typical structure of diseased plants) that contains spores that are spread by wind and of Sugar Cane Colonization. J. Fungi water [2], enhancing the dispersal of the pathogen. 2021, 7, 44. https://doi.org/10.3390 The plant induces several defensive mechanisms that determine the nature of the /jof7010044 interaction with the pathogen [3]. Similarly, pathogens develop mechanisms that enable them to evade and/or suppress the defensive responses of the plant [4,5]. The entry of Received: 9 December 2020 the infective mycelium into the vegetative bud meristem occurs within 6 to 36 h after Accepted: 9 January 2021 the teliospores have been deposited onto the bud scales [6]. Active penetration of fungal Published: 12 January 2021 hyphae from open stomata, ﬂoral organs, and even through the cuticle of the adaxial leaf epidermis have also been observed [7]. The cuticle can be mechanically destroyed as Publisher’s Note: MDPI stays neu- hyphae progress to the mesophilic layer of plants that are susceptible to infection [8]. tral with regard to jurisdictional clai- The subsequent growth of hyphae within the infected plant occurs mainly in the ms in published maps and institutio- parenchymatous cells of the lower internodes, achieving progress in its invasion by break- nal afﬁliations. ing the cell walls (Figure1). However, the pathogen can also remain in a dormant phase in the apoplastic area of the parenchymatous tissue [9]. Again, hyphae development concludes with the formation of the whip (teliospore sori) in the upper internodes.

Copyright: © 2021 by the authors. Li- It has been seen that different varieties of sugar cane manifest different responses censee MDPI, Basel, Switzerland. to an attack. For example, the Barbados (B) 42,231 cultivar (cv.) is highly susceptible to This article is an open access article infection. However, Mayarí (My) 55-14 is a resistant cv. that is able to defend itself against distributed under the terms and con- attack. Numerous studies have been directed toward explaining why sugar cane varieties ditions of the Creative Commons At- respond differently to smut colonization. However, what are the triggering mechanisms tribution (CC BY) license (https:// of infection? Herein, we present the way in which the fungus tries to infect plants in the creativecommons.org/licenses/by/ early stages. 4.0/).

J. Fungi 2021, 7, 44. https://doi.org/10.3390/jof7010044 https://www.mdpi.com/journal/jof J. FungiJ. Fungi 20212021, 7, ,7 x, 44FOR PEER REVIEW 2 of 18 2 of 19

1 mm

300 µm

FigureFigure 1. 1.( A(A) SEM) SEM micrograph micrograph of aof cross-section a cross-section of a healthyof a healthy sugar sugar cane leaf cane of leaf cv. Louisiana of cv. Louisiana 55-5. 55-5. ((BB)) SEM SEM micrograph micrograph of a of cross-section a cross-section of a smut-diseased of a smut-diseased leaf of sugar leaf caneof sugar cv. Louisiana cane cv. 55-5. Louisiana In this 55-5. In cut,this it cut, is possible it is possible to observe to observe some obturated some obturated xylem vessels xylem (red vessels arrow) (red and arrow) the subepidermal and the subepidermal and parenchymaland parenchymal tissue thattissue was that invaded was invaded by the fungal by the mycelium fungal (yellowmycelium arrow). (yellow arrow). 2. What Is the Life Cycle of the Ustilaginales-Like Sporisorium scitamineum? 2. What Is the Life Cycle of the Ustilaginales-Like Sporisorium scitamineum? First, to understand how the pathogen attacks, it is necessary to consider how its life cycleFirst, works. to understandS. scitamineum how(Syd), the pathogen previously attacks, known asit isUstilago necessary scitaminea to consider, is a basid- how its life iomycetecycle works. belonging S. scitamineum to the order (Syd), Ustilaginales, previously class known Ustilaginomycetes. as Ustilago scitaminea The life cycle, is ofa basidio- themycete smut belonging is simpler than to the that order of the Ustilaginales, rust since it is developedclass Ustilaginomycetes. on the same plant. The In life nature, cycle of the thesmut dikaryotic is simpler mycelium than that of these of the fungi rust seems since toit beis developed the cause of on its the infection same [ 10plant.,11]. Thus,In nature, the thedikaryotic primary mycelium mycelium isof saprophytic these fungi and seems of short to be duration, the cause with of its it notinfection being infectious [10,11]. Thus, the until it becomes dikaryotic through a process known as somatogamy. primary mycelium is saprophytic and of short duration, with it not being infectious until Although there are variations in the Ustilaginales’ cycles, there are some common characteristicsit becomes dikaryotic for the whole through group a (Figure process2A). known The cycle as somatogamy. includes, ﬁrst, the production of teliosporesAlthough within there the host are tissue variations [12]. After in maturing,the Ustilaginales’ the teliospores, cycles, which there are are gathered some in common thecharacteristics sori, undergo for karyogamy, the whole by whichgroup their (Figure two haploid2A). The nuclei cycle fuse includes, to form afirst, diploid the [ 13production], whileof teliospores acquiring awithin thick, darkthe host wall tissue that forms [12]. a blackishAfter maturing, powdery massthe teliospores, reminiscent which of smut. are gath- eredWhen in the the sori, conditions undergo are karyogamy, adequate, the by germination which their of two the teliosporeshaploid nuclei leads fuse to the to form a formationdiploid [13], of the while pro-mycelium acquiring thata thick, the nucleusdark wall moves that toward.forms a Thisblackish will thenpowdery undergo mass remi- meiosis,niscent originatingof smut. four haploid cells (sporidia or basidiospores), which can divide asex- ually via germination, originating a variable number of sporidia. The four sporidia that are initially released, as well as the cells formed from their mitotic division, are in no case pathogenic [12].

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B I

345 A 8 9 2

10 6

11 1 7

12 13

III

FigureFigure 2.2. ((AA)) Life Life cycle cycle of of UstilagoUstilago maydis maydis withwith the the stages stages represented represented as asfollows: follows: (1) teliospores (1) teliospores overwintering on soil, (2) germinating teliospore, (3) basidium, (4) basidiospores, (5) infection by overwintering on soil, (2) germinating teliospore, (3) basidium, (4) basidiospores, (5) infection by the basidiospores of young plants or growing tissues in older plants, (6) leaf infection by compati- the basidiospores of young plants or growing tissues in older plants, (6) leaf infection by compatible ble basidiospores, (7) galls on leaves, (8) corn ears infection by compatible basidiospores, (9) dikar- basidiospores,yotic mycelium (7) formation, galls on leaves, (10) mycelium (8) corn earsenlarges infection and byforms compatible galls in corn basidiospores, kernels, (11) (9) mycelium dikaryotic myceliumin galls, (12) formation, dikaryotic (10) cells mycelium of mycelium enlarges become and teliospores forms galls in galls, in corn and kernels, (13) galls (11) full mycelium of telio- in galls,spores. (12) (B dikaryotic) Differences cells in ofthe mycelium life cycle of become Sporisorium teliospores sp. as in represented galls, and (13)by (I) galls sugar full cane of teliospores. meriste- (maticB) Differences infection by in thebasidiospores, life cycle of (II)Sporisorium emergencesp. of the as representedwhip-like structur by (I)e sugar from canethe shoot meristematic apical infectionmeristem, by and basidiospores, (III) billions (II)of teliospores emergence produced of the whip-like in a single structure whip. from the shoot apical meristem, and (III) billions of teliospores produced in a single whip. When the conditions are adequate, the germination of the teliospores leads to the formationHowever, of the the pro-mycelium union of twoof that these the sporidia nucleus leads moves to thetoward. process This known will then as dikaryosis. undergo Dikaryoticmeiosis, originating hyphae are four infectious haploid and cells involves (sporidia forced or basidiospores), parasitism in nature which [ 10can,11 divide]. It presents asexu- aally polarized via germination, growth fromoriginating its extremities, a variable whichnumber is of typical sporidia. of filamentousThe four sporidia fungi that [7,14 are]. Theinitially dikaryotic released, mycelium as well growsas the insidecells formed the host from cells, their invading mitotic the division, whole plantare in and no pro-case ducingpathogenic the teliospores[12]. or ustilospores, which are gathered in great numbers, forming the so-calledHowever, sori. These the union teliospores of two orof sporesthese sporidia of the coals leads are to the the characteristic process known structures as dikaryosis. of the UstilaginalsDikaryotic hyphae and are are very infectious important and for involves their taxonomic forced parasitism classification. in nature They are[10,11]. formed It pre- en massesents a and polarized can develop growth at from different its extremities, places of the which host, is including typical of the filamentous flowers, leaves, fungi stems,[7,14]. rhizomes,The dikaryotic and in mycelium some cases, grows roots. inside the host cells, invading the whole plant and pro- ducingAs the in the teliospores rest of Ustilaginales, or ustilospores, the diploidal which are and gathered dikaryotic in great mycelium numbers, of S. scitamineumforming the isso-called the one sori. that These has infective teliospores capacity or spores since of it the manages coals are to penetratethe characteristic the host structures tissues and of damagethe Ustilaginals the meristematic and are very tissues important of the plant for their [15]. taxonomic However, classification. despite the evident They are proximity formed between Sporisorium sp. and Ustilago sp., these present certain differences in their modes en masse and can develop at different places of the host, including the flowers, leaves, of infection (Figure2B). Ustilago infects the aerial parts of the plant [16], rapidly forming stems, rhizomes, and in some cases, roots. gills or tumors full of teliospores, while Sporisorium sp. infects young seedlings and can As in the rest of Ustilaginales, the diploidal and dikaryotic mycelium of S. scitami- remain asymptomatic and progresses systemically [17]. In the case of this pathogen, the life neum is the one that has infective capacity since it manages to penetrate the host tissues and cycle ends with the emergence of the whip-like structure from the shoot apical meristem, damage the meristematic tissues of the plant [15]. However, despite the evident proximity where billions of the teliospores that are produced in a single whip are easily dispersed in between Sporisorium sp. and Ustilago sp., these present certain differences in their modes the field by wind, rain, and small animals [16]. of infection (Figure 2B). Ustilago infects the aerial parts of the plant [16], rapidly forming

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At this point, it is interesting to note that infection starts again when some teliospores are deposited into other plants. As such, how does the pathogen “make sure” that a new infection will be successful?

3. A Quorum Signal (QS) Triggers the Infection To ensure an efficient attack, the size of the teliospore colony on a leaf’s surface, the buds, or a stem must reach a critical mass that puts up sufficient resistance to the possible defense mechanisms that the plant triggers in its presence. In other words, the pathogen must develop cell aggregation mechanisms such that enough infective cells survive the attack of the plant through the plant’s resistance and defense factors, i.e., “unity makes strength.” How do fungal cells communicate with each other? Pathogenic and symbiotic bacteria depend substantially on quorum signals (QSs) to colonize and infect their hosts [18,19]. In fact, it has been seen that open stomata are initially colonized by a few bacteria, rapidly increasing the population of the pathogen over time on that structure. QSs are a system that regulates gene expression in bacteria and is dependent on the population density [20]. This enables individual bacterial cells in a local population to coordinate the expression of certain genes, helping them to behave in a similar way to a multicellular organism. QSs work via an exchange of small signal molecules between nearby bacteria. The cell population increases as a function of the signal concentration. Operationally, a bacterial quorum is present when the signal concentration reaches levels that are capable of triggering changes in the gene expression. QS is particularly important for the ability to infect the plant with pathogenic bacteria. Defective mutants in QSs are avirulent or show very reduced virulence [21]. Extracellular autoinducing QS molecules in the supernatants of microbial cultures were first recognized for their roles in the induction of genetic competence in Gram-positive bacteria [22]. However, very recently, it has been discovered that fungi can also develop QS signaling systems that affect not only the population size, but also the morphology, biofilm formation, and pathogenicity [23,24]. It seems that the common mechanism of the quorum involves the synthesis of signals that are released outside the cell via active transport or diffusion [24]. Therefore, intraspecific communication is possible because of the response to QS molecules that pathogens accumulate in their extracellular environment [22]. The natures of these QS molecules vary enormously: lactones, cyclic dipeptides, and methyl esters of fatty acids have all been described as QS inductors. Candida albicans, a dimorphic fungus, was the first studied as having a QS system [22]. C. albicans transitions from a yeast-like growth to polarized filaments, where this capacity seems to be essential in the diffusion of the disease. Hyphae formation is spontaneously suppressed for high cell densities or when culture media are added to supernatants of cultures in the stationary growth phase. This suggested that hyphae formation is partially regulated by some soluble and diffusible factors [22]. Hornby et al. [25] identified this signal molecule as farnesol, which is active against many strains of C. albicans in a concentration range from 1 to 50 µM. Apart from farnesol, other QS molecules in C. albicans are the aromatic amino-acid-derived alcohols like tyrosol, tryptophol, and phenylethanol [26]. Other fungi have been shown to produce extracellular molecules that modulate cell morphology. Uromyces phaseoli produce methyl-3,4-dimethoxycinnamate, an autoinhibitor of the germination of its own spores that is effective at nanomolar concentrations [27]. In Glomerella cingulata cultures, a diffusible factor decreases mycelium formation with a con- comitant increase in conidia formation at cell densities above 106 cells mL−1. The chemical identity of this molecule has not yet been described [28]. Many times, QS molecules cause variations in the regulation of gene expression as a response to cell density [29] to promote invasion under optimal conditions. Interestingly, Vitale et al. [30] reveal the role of peptide pheromones in cell density regulation in Fuxar- ium oxysporum development. In a similar way, Sanchez-Elordi et al. [31] have shown that S. scitamineum spores in an aqueous medium secrete a glycosylated enzyme with arginase activity that binds to wall receptor points that are similar or identical to those described by J. Fungi 2021, 7, x FOR PEER REVIEW 5 of 19

Many times, QS molecules cause variations in the regulation of gene expression as a response to cell density [29] to promote invasion under optimal conditions. Interestingly, Vitale et al. [30] reveal the role of peptide pheromones in cell density regulation in Fux- J. Fungi 2021, 7, 44 arium oxysporum development. In a similar way, Sanchez-Elordi et al. [31] have shown5 ofthat 18 S. scitamineum spores in an aqueous medium secrete a glycosylated enzyme with arginase activity that binds to wall receptor points that are similar or identical to those described by Millanes et al. [32] and interact with sugarcane defense proteins. This arginase seems Millanesto have different et al. [32 domains] and interact of interaction with sugarcane with these defense ligands proteins. in such This a way arginase that the seems enzyme to havecan adhere different to domainsone or several of interaction teliospores, with producing these ligands a cytoagglutination in such a way that effect. the enzyme Cytoagglu- can adheretination to has one been or several described teliospores, as a quorum producing signal a since cytoagglutination the S. scitamineum effect. arginase Cytoagglutina- acceler- tionates hasthe beengermination described of the as a group quorum when signal it binds since to the theS. scitamineumteliospore cellarginase wall [33]. accelerates Figure 3 theshows germination the aggregation of the group of teliospores when it bindsover time. to the These teliospore images cell were wall obtained [33]. Figure after3 shows the in- the aggregation of teliospores over time. These images were obtained after the incubation cubation of teliospores in Lilly–Barnett medium from 0 to 72 h. During this time, the of teliospores in Lilly–Barnett medium from 0 to 72 h. During this time, the spores began spores began to germinate and produce arginase. to germinate and produce arginase. Time

0h 24h 48h 72h

A B C D

10 µm 10 µm 10 µm 10 µm FigureFigure 3.3. QuorumQuorum sensingsensing ofof smutsmut teliosporesteliospores viavia thethe actionaction ofof itsits ownown arginasearginase thatthat waswas secretedsecreted intointo thetheLilly–Barnett Lilly–Barnett medium. The degree of cytoagglutination was displayed after 0 h (A), 24 h (B), 48 h (C), and 72 h (D) of incubation. medium. The degree of cytoagglutination was displayed after 0 h (A), 24 h (B), 48 h (C), and 72 h (D) of incubation.

QuorumQuorum signalingsignaling isis suchsuch aa relevantrelevant processprocess thatthat inin recentrecent years,years, manymany studiesstudies havehave beenbeen directeddirected toto elucidateelucidate thethe molecularmolecular mechanismmechanism thatthat inhibitsinhibits thethe QSQS inin fungifungi inin orderorder toto controlcontrol pathogenpathogen developmentdevelopment [[24].24]. However,However, itit isis interestinginteresting thatthat whatwhat sciencescience seeksseeks cancan be found already already in in nature. nature. For For example, example, it ithas has been been described described that that resistant resistant My My 55- 55-1414 sugar sugar cane cane plants plants simulate simulate a quorum a quorum signal signal using using their their own own arginase, arginase, where where the pro- the productionduction of ofthis this enzyme enzyme is isstrongly strongly increased increased by by the the inoculation inoculation of of healthy plantsplants withwith smutsmut sporidia.sporidia. CytoagglutinationCytoagglutination seems toto bebe necessarynecessary toto traptrap thethe teliosporesteliospores inin aa smallsmall regionregion ofof thethe spacespace inin contactcontact withwith canecane arginase,arginase, whichwhich wouldwould increaseincrease theirtheir accessibilityaccessibility toto the cell walls walls and and facilitate facilitate the the enzymatic enzymatic activities activities of ofcane cane glycoproteins glycoproteins to degrade to degrade the thetrapped trapped teliospores teliospores [33]. [33 Theref]. Therefore,ore, an efficient an efﬁcient false false quorum quorum signal, signal, only only produced produced by byresistant resistant varieties, varieties, appears appears to be to essential be essential for the for plant the plant to simultaneously to simultaneously attack attack the max- the maximumimum number number of fungal of fungal cells cells

4.4. Pathogenicity FactorsFactors PhytopathogenicPhytopathogenic bacteriabacteria andand fungifungi havehave secretionsecretion systemssystems throughthrough whichwhich theythey cancan injectinject virulencevirulence effectorseffectors intointo thethe hosthost cells;cells; thesethese effectorseffectors areare generallygenerally proteinsproteins butbut alsoalso smallsmall moleculesmolecules thatthat displaydisplay variedvaried actions on the cellular machinery. Up to thethe present,present, severalseveral of these these secreted secreted molecules molecules have have been been described described that, that, generalizing generalizing the theconcept, concept, are arealso also defined deﬁned as virulence as virulence factors, factors, given given their their absolute absolute requirement requirement to inject to inject into the into plant the plantto develop to develop disease disease [34]. [34]. S.S. scitamineumscitamineum possess a diverse range range of of effector effectorss that that are are directed directed toward toward manipu- manip- ulating the host metabolism [21,35,36] and to defending itself from the plant’s immune lating the host metabolism [21,35,36] and to defending itself from the plant’s immune sys- system. From active-growing mycelium S. scitamineum, a group of proteins that develop a tem. From active-growing mycelium S. scitamineum, a group of proteins that develop a biological action on the host tissues has been isolated via extraction in aqueous medium biological action on the host tissues has been isolated via extraction in aqueous medium and penetrability chromatography [37]. The most active fraction (fraction 5) only contained protein, while the rest of the fractions, of which the most active were 6 and 7, were glycoproteins. After incubating the leaf discs of the smut-sensitive Barbados (B) 42231 cv. and the resistant Mayarí My 55-14 cv. in solutions of these fractions, it was observed that the total phenol content was similar in the control discs of untreated and treated plants at time zero, but it increased notably after the incubation of the discs with the protein of fraction 5, and to a lesser extent, although very signiﬁcantly, with the glycoproteins contained in fractions 6 and 7. The varietal difference implies a greater accumulation of free phenols in J. Fungi 2021, 7, x FOR PEER REVIEW 6 of 19

and penetrability chromatography [37]. The most active fraction (fraction 5) only contained protein, while the rest of the fractions, of which the most active were 6 and 7, were glycoproteins. After incubating the leaf discs of the smut-sensitive Barbados (B) 42231 cv. and the resistant Mayarí My 55-14 cv. in solutions of these fractions, it was observed that the total phenol content was similar in the control discs of untreated and treated plants at time zero, but it increased notably after the incubation of the discs with the protein of fraction 5, and to a lesser extent, although very significantly, with the glycoproteins contained in fractions 6 and 7. The varietal difference implies a greater accumulation of free phenols in the leaves of the sensitive cultivar. This increase somehow corresponds to the J. Fungi 2021, 7, 44 activation of the phenylalanine ammonia lyase (PAL) enzyme, which6 of 18is higher in My 55- 14 than in B 42231, as well as the peroxidase (POX) system. The lower PAL activity in the sensitive cultivar could be related to the levels of caffeic acid and its derivative, chloro- thegenic leaves acid, of the which sensitive remain cultivar. high This in increase cv. B 42231 somehow but corresponds not in cv. toMy the 55-14. activation The accumulation of of the phenylalanine ammonia lyase (PAL) enzyme, which is higher in My 55-14 than in Bcaffeic 42231, asacid well will as the then peroxidase produce (POX) a feedback system. The inhibi lowertion PAL of activity the PAL in the such sensitive that its detectable ac- cultivartivity levels could be in related the sensitive to the levels varieties of caffeic would acid and be its lower. derivative, This chlorogenic would indicate acid, that virulence whichfactors, remain i.e., highboth in proteins cv. B 42231 and but glycoproteins, not in cv. My 55-14. would The accumulation activate phytoalexin of caffeic synthesis in the acid will then produce a feedback inhibition of the PAL such that its detectable activity levelssensitive in the cultivar sensitive varieties(phenols would derived be lower. from This wouldhydroxycinnamic indicate that virulence acids, factors, such as flavonoids or i.e.,tannins), both proteins while and in glycoproteins, the resistant would cultivar, activate phenols phytoalexin derived synthesis from in the sensitivea PAL activity would be cultivarpreferentially (phenols deriveddirected from toward hydroxycinnamic lignin and acids, lignan such synthesis, as ﬂavonoids in orwhich tannins), peroxidases play an whileessential in the resistantrole (Figure cultivar, 4). phenols In this derived sense, from the a PAL production activity would of belignans preferentially has been considered a directed toward lignin and lignan synthesis, in which peroxidases play an essential role (Figuredefense4). factor In this sense,of the the plant, production in addition of lignans to hasthe been production considered and a defense secretion factor of defense proteins of[38]. the plant,In fact, in addition the way to thein productionwhich sugar and secretioncane plants of defense direct proteins their [38 resources]. In fact, to lignin/lignan thesynthesis way in which by means sugar caneof the plants regulation direct their of resources dirigent to protein lignin/lignan levels synthesis seems by to be critical in the means of the regulation of dirigent protein levels seems to be critical in the early defense of resistantearly defense My 55-14 of cvs. resistant [38,39]. My 55-14 cvs. [38,39].

NH3

COOH COOH PAL Cinnamic a cid Phenylalanine NH2 Cinnamate-4- -hydroxylase O2

HO HO COOH COOH Caffeic acid p-Coumarate-3- p-Coumaric acid HO -hydroxylase SAM SAM-cafeoyl-3-O- methyl transferase Cytochrome c P450 SAHC feruloyl-5-hydroxylase

SAM feruloyl-5-O- methyl transferase HO HO COOH CH2OH Feruloyl-CoA ligase Ferulic acid Coniferyl alcohol CH O CH3O 3 Feruloyl-CoA:NADPH oxido-reductase Cinnamoyl alcohol dehydrogenase

Peroxidases HO Laccases

CH O 3 O OCH3 O

OH LIGNINS (+)-pinoresinol DIRIGENT PROTEINS

(-)-pinoresinol LIGNANS FigureFigure 4. 4.Caffeic Caffeic acid acid acts acts as a regulatoras a regulator of the initiation of the initiation of lignin biosynthesis of lignin biosynthesis via the feedback via the feedback inhibitioninhibition of phenylalanineof phenylalanine ammonia ammonia lyase in smut-sensitive lyase in smut-sensitive cultivars. cultivars.

J. Fungi 2021, 7, 44 7 of 18

In vitro studies evidence that some proteins related to cell wall modification, morphogenesis, polysaccharide degradation, and carbohydrate metabolism are exclusively secreted in response to host extract media, probably at early time points during the penetration and colonization of sugarcane cells [40]. Recently, Teixeira-Silva et al. [41] have detected S. scitamineum effectors, which are critical molecules that are be able to defeat sugar cane defense mechanisms. Various potential interactors were identified, including subunits of the protein phosphatase 2A and an endochitinase. The results evidence that the expression of effectors is influenced by the sugarcane genotypes during tissue colonization. Moreover, it seems that orthologs of sugarcane that share around 70% similarity may be the plant targets of these effectors. Some species of corn smut, such as U. maydis, have genes coding for virulence factors that are secreted into the environment. Interestingly, the S. scitamineum sequenced genome contains more genetic clusters or groups for these secreted effectors than other similar species [36]. This increase in the number of genes appears to be due to tandem genetic duplication and to the existence of other elements that are associated with these genes [42]. Some of these genes are related to the synthesis of enzymes that are responsible for the degradation of the cell wall, and because of that, they are clearly involved in the development of the virulence of the pathogen [36]. During the coevolution of fungal plant pathogens and their hosts, a seesawing in- terplay between pathogen virulence and host resistance has been developed. It is an interesting point of plant–pathogen coevolution that the early defense of sugar cane is also focused on cell wall degradation. Enzymes, such as chitinases [43], glucanases, and peroxidases, and sometimes catalases [44], have been traditionally related to the defense mechanisms of the plant. In the same way, polyamine accumulation in S. scitamineum, at moderate levels, is considered a virulence factor that is necessary for smut growth and pathogenicity [45] as in other Ustilaginales [46]. In U. maydis, putrescine is an essential molecule for the transformation into infective mycelium, as sporidia that are unable to produce putrescine cannot achieve the transition to the dimorphic state [47]. The formation and growth of dikaryotic hyphae after sexual mating is critical for S. scitamineum’s pathogenicity; Chang et al. [48] demonstrated that an elevated intracellular ROS (reactive oxygen species) level promotes S. scitamineum mating filamentation via the transcriptional regulation of ROS catabolic enzymes and is regulated by the cAMP/PKA signaling pathway. A connection between putrescine production and ROS has been suggested [49,50], where moderated levels of that polyamine may be involved in sporidia transformation into dikaryotic mycelium. However, increased production of these molecules induced by sugarcane glycoproteins leads to nuclear decondensation, cell wall breakdown, and germinative blockage of teliospores as a part of the plant’s defense mechanism [45]. It seems that the germination blocking by means of a polyamine level increase occurs through the disruption of the cytoskeleton. Moreover, the role of sugar cane arginase (the one that is responsible for false quorum signals) in cytoskeletal disorganization has been amply studied [50,51]; as such, how important is an organized cytoskeleton in teliospores for pathogenicity and development?

5. The Role of the Cytoskeleton in Teliospore Germination Fuchs et al. [52] showed that F-actin is essential for polarized growth during the infection of corn with U. maydis, a pathogen that is phylogenetically related to S. scitamineum. In addition, through their role in cytoskeleton organization, Rho GTPases are required for the establishment and maintenance of cell polarity, as well as polarized growth in fungal cells [53]. Numerous experiments in the presence of the inhibiting agents of cytoskeletal functionality have demonstrated the involvement of actin in the establishment of cell polarity. Incubation with latrunculin A, a depolymerizing agent of actin ﬁlaments [54], leads to depolymerization of the ﬁlaments in Saccharomyces cerevisae cells, compromising their sporulation [55]. J. Fungi 2021, 7, x FOR PEER REVIEW 8 of 19

J. Fungi 2021, 7, 44 8 of 18 The main cellular event that precedes the germination of smut teliospores is the establishment of cell polarity [56]. These results are in agreement with those obtained by The main cellular event that precedes the germination of smut teliospores is the es- tablishmentBachewich of and cell polarityHeath [57], [56]. Thesewho demonstrated results are in agreement that F-actin with those participates obtained byin such polariza- Bachewichtion and andin the Heath beginning [57], who of demonstrated the protru thatsion F-actin of the participates end of a innascent such polariza- hypha in Saprolegnia tionferax and. Millanes in the beginning et al. [58] of found the protrusion a total absence of the end of of cell a nascent polarization hypha ininSaproleg- non-germinated, rest- niaing ferax teliospores,. Millanes et as al. revealed [58] found by a total the absenceabsence of of cell staining polarization of F-actin in non-germinated, with fluorescein isothio- restingcyanate teliospores, (FITC)-labeled as revealed phalloidin by the absence (Figure of staining 5A), ofbut F-actin G-actin with molecules ﬂuorescein isothio- began to polymerize cyanate (FITC)-labeled phalloidin (Figure5A), but G-actin molecules began to polymerize andand polarize polarize after after teliospore teliospore incubation incubation in Lilly–Barnett in Lilly–Barnett medium (Figure medium5B). After (Figure staining 5B). After stain- withing FITC-phalloidin,with FITC-phalloidin, actin capping actin marked capping the site mark on theed teliospore the site wallon throughthe teliospore which wall through thewhich germ the tube germ emerged tube (Figure emerged5C). (Figure 5C).

FigureFigure 5. 5.Polarization Polarization of the of F-actin the F-actin microfibrils microfibrils and appearance and a ofppearance capping after of capping the incubation after of the incubation of teliosporesteliospores with with fluorescein fluorescein isothiocyanate isothiocyanate (FITC)-phalloidin. (FITC)-phalloidin. (A) Resting cells (A in) Resting which capping cells didin which capping notdid appear. not appear. (B) Initiation (B) Initiation of capping of (arrows) capping after (arrows) a few hours after of a rehydration. few hours (ofC) rehydration. A germinated (C) A germi- spore,nated with spore, the hyphawith the emerging hypha from emer theging teliospore from through the teliospore the germinative through pore, the as signalizedgerminative by pore, as signal- theized capping. by the The capping. dark zone The opposite dark tozone this opposite germination to point this cangermination be observed point (arrow). can be observed (arrow). The cytoplasm polarization marked by the formation of F-actin microfibrils is pre- ventedThe by the cytoplasm use of several polarization inhibitors, such marked as phalloidin, by the an formation actin cytoskeleton of F-actin stabilizer microfibrils is pre- thatvented prevents by the the use depolymerization of several inhibitors, of F-actin such [59], latrunculinas phalloidin, A, which an actin is a chemical cytoskeleton stabilizer agentthat prevents that keeps the actin depolymerization depolymerized [54], of or blebbistatin,F-actin [59], which latrunculin is a myosin A, IIwhich con- is a chemical tractilityagent that inhibitor keeps [60 actin]. In depolymerized addition, several glycoproteins[54], or blebbistatin, produced which by the hostis a myosin plant, II contractil- particularly that which exhibits arginase activity, can inhibit actin capping and, therefore, preventity inhibitor teliospore [60]. germination. In addition, several glycoproteins produced by the host plant, particu- larlyThe that organization which exhibits of the actin argi innase the early activity, stages of can the inhibit pathogen’s acti lifen cyclecapping (Figure and,6B) therefore, pre- confirmedvent teliospore that an organized germination. distribution of F-actin should be a key condition for the proper progressThe of sporidiaorganization emergence of the [51 ].actin In this in contribution, the early st informationages of the gathered pathogen’s from the life cycle (Figure micrographs obtained by confocal microscopy made it possible to draw up a scheme of the6B) organization confirmed of that the an microfilaments organized duringdistribution the early of stages F-actin of development.should be a F-actinkey condition for the andproper myosin progress were jointly of sporidia located in emergence situ in the teliospores [51]. In duringthis contribution, the first stages information of their gathered germination,from the micrographs as can also be obtained seen in Figure by 6confocalA. It was foundmicroscopy that actin made and myosinit possible are to draw up a scheme of the organization of the microfilaments during the early stages of development. F-actin and myosin were jointly located in situ in the teliospores during the first stages of their germination, as can also be seen in Figure 6A. It was found that actin and myosin are distributed throughout the cellular cytoplasm (including the cortical zone in the case of actin) in the teliospores. Both proteins co-localize inside the cell, which suggests that they act together. After the emergence of hyphae, it was observed that actin marking tended to disappear in the teliospore (mainly in the cortical region), intensifying in the cytoplasm of

J. Fungi 2021, 7, 44 9 of 18

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actin) in the teliospores. Both proteins co-localize inside the cell, which suggests that they act together. After the emergence of hyphae, it was observed that actin marking tended to thedisappear germinative in the tube, teliospore whose (mainly growth in was the then cortical active region), [51]. intensifyingThus, the teliospores in the cytoplasm transport of newthe germinativematerial in the tube, direction whose growthof the end was of then the active nascent [51 ].hyphae, Thus, theas occurs teliospores commonly transport in othernew materialfilamentous in the fungi direction [14]. However, of the end while of the the nascent cellular hyphae, content as is occurs translocated commonly to the in interiorother ﬁlamentous of the emerging fungi germinative [14]. However, tube, while the teliospore the cellular loses content its function is translocated and degener- to the interior of the emerging germinative tube, the teliospore loses its function and degenerates, ates, thus gradually diluting the F-actin mark. thus gradually diluting the F-actin mark.

FigureFigure 6. (A) ImageImage obtainedobtained using using confocal confocal microscopy microscopy that that displays displays to ato group a group of teliospores. of teliospores. The greenThe green color color corresponds corresponds to Alexa-Fluor®-488-conjugated phalloidin labeling. The red color corresponds to labeling with an anti-phosphor- to Alexa-Fluor®-488-conjugated phalloidin labeling. The red color corresponds to labeling with an anti-phosphorylated ylated MLC-Ser19 antibody. Scale bars indicate 2 µm. (B) Series of images obtained using confocal microscopy that dis- MLC-Ser19 antibody. Scale bars indicate 2 µm. (B) Series of images obtained using confocal microscopy that displays a plays a germinating teliospore labeled with Alexa-Fluor®-488-conjugated phalloidin. Scale bars indicate 2 µm. germinating teliospore labeled with Alexa-Fluor®-488-conjugated phalloidin. Scale bars indicate 2 µm. Microtubules (MTs), as actin filaments, are also involved in S. scitamineum germina- Microtubules (MTs), as actin filaments, are also involved in S. scitamineum germina- tiontion [50]. [50]. While While latrunculin latrunculin (an (an inhibitor inhibitor of actin of actin polymerization polymerization [54]) [ 54blocks]) blocks sporidia sporidia for- mationformation and and therefore therefore does does not notallow allow germinatio germinationn to begin, to begin, nocodazole nocodazole (a drug (a drug responsible responsi- forble MTs for MTs disorganization disorganization [61]) [61 completely]) completely prev preventsents sporidia sporidia release release but but not not germinative germinative tubetube formation. formation. In In this this case, case, germinative germinative tubes tubes are are formed formed but but they they cannot cannot be be released released untiluntil the the nucleus nucleus reaches reaches the the body body of ofthe the hyph hyphae;ae; sporidia sporidia liberation liberation must must “wait” “wait” for nu- for cleusnucleus repositioning. repositioning. Therefore, Therefore, MTs MTsin S. inscitamineumS. scitamineum are notare necessary not necessary for hyphae for hyphae elongation,elongation, but they but may they be may involved be involved in the innuclea the nuclearr repositioning repositioning of the ofgrowing the growing hyphae. hyphae. Such mechanisticSuch mechanistic separation separation has been has ob beenserved observed in other in othersystems systems [62–64]. [62 –64]. TheThe presence presence of of nuclear nuclear migration migration with with the the help help of of the the cytoskeleton cytoskeleton during during germina- germina- tiontion is is supported supported by by other other experiments. experiments. Th Thee use use of of the the fluorescent fluorescent antibodies anti- anti-αα-actin-actin andand anti-tubulin anti-tubulin has has allowed allowed for for obtaining obtaining micrographs micrographs using using a a confocal confocal microscope microscope that that clearlyclearly indicates indicates the simultaneous presence of bothboth polymericpolymeric systems,systems, namely,namely, microfib-microfi- brilsrils andand MTsMTs (Figure (Figure7 ,7, series series 1 1 and and 3), 3), which which were were bothboth inin thethe nucleinuclei ofof the teliospores and werewere revealed revealed by by means means of of staining staining with with 4.6-diamino-2-phenylindol 4.6-diamino-2-phenylindol (DAPI), (DAPI), and and in the in cy- the toplasmcytoplasm of ofthe the hypha hypha in inactive active growth growth (Figure (Figure 7,7 ,series series 2 2 and and 3). 3). The The coexistence coexistence of bothboth systems,systems, namely, namely, microfibrils microfibrils and microtubules, clearly indicates that the nucleus nucleus can can move move inin a a directed directed way throughthrough thethe actively actively growing growing hyphae hyphae once once the the teliospore teliospore has germinated,has germinated,as is presented as is presented in previous in previous works works [50]. [50].

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Brightfield α-actin DAPI Merged

N 1 T

Brightfield α-actin DAPI Merged

N S S N S 2 S N N

Brightfield α-tubulin DAPI Merged N

S S T S T N H H 3 S N

FigureFigure 7. 7. ConfocalConfocal series series (z-maxprojection (z-maxprojection)) of cells. Brightfield, Brightfield, DAPI, αα-actin-actin images,images, asas wellwell asas brightfield/DAPI/brightfield/DAPI/αα-actin-actin composites,composites, are are shown shown in the 1 an andd 2 series. Brightfield,Brightfield, DAPI, α-tubulin-tubulin images,images, asas wellwell asas brightfield/DAPI/ brightfield/DAPI/α-tubulin-tubulin composites, are shown in series number 3 (N—nucleus; H—hyphae; S—sporidia; T—teliospore). Scale bars indicate 2 µm. composites, are shown in series number 3 (N—nucleus; H—hyphae; S—sporidia; T—teliospore). Scale bars indicate 2 µm.

6.6. The The Role Role of of the the Cytoskeleton Cytoskeleton in in Teliospore Teliospore Motility Motility BecauseBecause the the pathogen pathogen can can use use the the open open stomata stomata and and other other openings openings of of the the host host as as a a routeroute of of entry entry to to penetrate penetrate the the internal internal tissues tissues [7], [7], it it is is thought thought that that teliospores teliospores deposited deposited atat random random on on a aleaf’s leaf’s surface surface should should develo developp mechanisms mechanisms of displacement of displacement toward toward the en- the tryentry pathways pathways to the to plant. the plant. Brand Brand and Gow and [6 Gow5] summarize [65] summarize the current the currentknowledge knowledge regard- ingregarding spore movement spore movement during during plant–pathogen plant–pathogen interaction. interaction. The two The most two mostfrequently frequently sug- gestedsuggested mechanisms mechanisms are submicroscopic are submicroscopic contractions contractions of helically of helically distributed distributed fibrils in fibrils cell wallsin cell and walls the andexistence the existence of mobile of appendages mobile appendages in zoospores. in zoospores. Other species Other of pathogenic species of fungipathogenic produce fungi spores produce that sporescan move that via can a movemechanism via a mechanism called gliding. called This gliding. type of This move- type mentof movement differs from differs dragging from dragging or swimming or swimming in that in gliding that gliding does doesnot involve not involve the theaction action of anyof any apparent apparent external external motility motility or any or obvious any obvious change change in cell size, in cell and size, furthermore, and furthermore, always requiresalways requiresthe presence the presence of a substrate. of a substrate. The cytoskeleton The cytoskeleton of S. scitamineum of S. scitamineum cells is alsocells in- is volvedalso involved in the movement in the movement of cells of to cells a chemotherapeutic to a chemotherapeutic agent produced agent produced by the byhost the plant host [66].plant Numerous [66]. Numerous models models of chemotaxis of chemotaxis have havebeen beendescribed described regarding regarding plant–pathogen plant–pathogen interactions,interactions, where where motility motility is an is important an important feature feature of virulence of virulence [67]. [67 ]. ChemotaxisChemotaxis in in eukaryotes eukaryotes involves involves the the diff differentiationerentiation of of two two cellular cellular poles poles during migration:migration: one one directed directed toward toward the the source source of of the the chemoattractant, chemoattractant, in in favor favor of of the the gradient, gradient, andand the the other other in in the the opposite opposite direction. direction. These These poles poles show show differences differences in the in the distribution distribution of theirof their components, components, specifically specifically those those involved involved in the in signaling the signaling and rearrangement and rearrangement of cyto- of skeletoncytoskeleton proteins proteins in plants in plants and and animals animals [68,69]. [68,69 ].Thus, Thus, during during chemotaxis, chemotaxis, the the establish- establish- mentment of of a a cellular cellular asymme asymmetrytry constitutes constitutes the the basis basis of of polarized polarized movement. movement. ExperimentsExperiments carried carried out out by by Sánc Sánchez-Elordihez-Elordi et al. et al.[66] [ 66evaluated] evaluated the migration the migration of teli- of osporesteliospores toward toward sugarcane sugarcane glycoproteins. glycoproteins. Curi Curiously,ously, migration migration was was more more efficient efficient after after contactcontact with with glycoproteins glycoproteins from from resistant resistant pl plants,ants, which which suggests suggests that that induced induced chemoat- chemoat- tractiontraction is is an an early early resistance resistance mechanism mechanism that that is is probably probably directed directed to to enhance enhance the the false false quorumquorum signal [[33,50,51].33,50,51]. OnOn the the other other hand, hand, the the displacement displacement of S. of scitamineum S. scitamineumteliospores telio- sporeswas radically was radically diminished diminished in the in presence the presence of inhibitors of inhibitors of cytoskeleton of cytoskeleton organization organization (la- (latrunculin A, phalloidin, and blebbistatin), which confirmed that the cytoskeleton must

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betrunculin directly A, involved phalloidin, in andthe motility blebbistatin), of the which teliospores conﬁrmed toward that thethe cytoskeleton chemo-attracting must agent. Micrographsbe directly involved obtained in the using motility SEM of and the TEM teliospores (Figur towarde 8) demonstrate the chemo-attracting the absence agent. of external structuresMicrographs related obtained to motility using SEM in andteliospores TEM (Figure that8 )were demonstrate previously the absence exposed of externalto the chemoat- structures related to motility in teliospores that were previously exposed to the chemoattractant (Figure 8A,B). However, the presence of invaginations, which were clearly visible, tractant (Figure8A,B). However, the presence of invaginations, which were clearly visible, inin one one of of the the cellular polespoles stands stands out out (Figure (Figur8eC). 8C). It has It has been been hypothesized hypothesized that thesethat these in- vaginationsinvaginations arose arose because because ofof cytoskeletal cytoskeletal reorganization reorganization to direct to direct the cell the toward cell toward the the chemoattractantchemoattractant in in a a liquid liquid medium medium or, ator, least, at least, on solid on solid surfaces surfaces (leaves, (leaves, stalks) coveredstalks) covered byby drops drops ofof condensedcondensed water water [66 [66].].

FigureFigure 8. SEMSEM micrographsmicrographs of of (A ()A a) germinated a germinated teliospore teliospore and (andB) aggregated (B) aggregated teliospores teliospores after 5 hafter 5 h ofof chemotactic chemotactic stimulation with with sugar sugar cane cane glycoproteins glycoproteins from from the the resistant resistant My 55-14My 55-14 cv. show- cv. showing superficial,ing superﬁcial, non-motile non-motile appendages. appendages. ( (CC)) TEM TEM micrograph of of a teliosporea teliospo thatre that was was chemotactically chemotactically stimulatedstimulated with with sugar sugar cane cane glycoproteins glycoproteins from from the resistant the resistant My 55-14 My cv., 55-14 where cv., the where white arrowthe white indi- arrow indicatescates the invaginated the invaginated pole opposite pole opposite the emergence the emerge pointnce of the point nascent of the hypha. nascent This zone,hypha. opposite This zone, to op- positethe invaginated to the invaginated pole, would pole, mark would the advance mark frontthe ad ofvance the teliospore front of in the a liquid teliospore medium. in a liquid medium.

7.7. The The GTPases as as Mediators Mediators of of the the Signal Signal of Organization of Organization of the of Cytoskeleton the Cytoskeleton When circumstancescircumstances become become favorable, favorable, processes processes of chemoattraction,of chemoattraction, cytoaggluti- cytoagglutina- nation by QS molecules, and germination are induced in the S. scitamineum population. tion by QS molecules, and germination are induced in the S. scitamineum population. Therefore, the cytoskeletal reorganization of teliospores is the result of a speciﬁc and con- Therefore,trolled response the cytoskeletal to environmental reorganization conditions. of GTPases teliospores have traditionallyis the result beenof a specific described and con- trolledas the link response between to the environmental reception of an conditions external signal. GTPases and its have transduction traditionally inside been the cell described asto the produce link between a physiological the reception response of [70 an]. external Thus, the signal Rho GTPases and its acttransduction like switches inside of large the cell to producesignaling a cascades physiological by alternating response their [70]. active Thus, and inactivethe Rho states, GTPases binding act tolike GTP switches or GDP, of large signalingrespectively cascades [71]. The by signals alternating are often their derived active fromand inactive extracellular states, ligands binding that to activate GTP or GDP, respectivelyguanosine nucleotide [71]. The exchange signals factors are often (GEFs) deriv that catalyzeed from the extracellular replacement ofligands GDP by that GTP activate guanosine nucleotide exchange factors (GEFs) that catalyze the replacement of GDP by GTP in the GTPases, allowing their activation. Only in their active conformation, when bound to GTP, can Rho proteins interact with specific effectors inside the cell [72]. Among all the possible intracellular responses, their activity directly influences the organization of the cytoskeleton [73].

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in the GTPases, allowing their activation. Only in their active conformation, when bound to GTP, can Rho proteins interact with specific effectors inside the cell [72]. Among all the possible intracellular responses, their activity directly influences the organization of the cytoskeleton [73]. The organization of the cytoskeleton in S. scitamineum cells is also a consequence of a signaling cascade triggered by the action of GTPase proteins [66]. The addition of GTP to the teliospores’ incubation medium partially reverses the inhibitory effect of latrunculin A, probably through Rho activation; in contrast, neither the addition of GTPγS, a non- hydrolyzable analog of GTP that keeps GTPase activated [74], nor the addition of GDPβS, a deactivator of Rho GTPases [75], were able to reverse the inhibitory effect. These results indicate that GTPγS may behave as a strong Rho activator such that cells may respond to this hyperactivation by inactivating the pathway. This suggests that Rho proteins should play an important role as a communication axis in the production and modulation of cellular responses to different forms of stress [76]. On the other hand, GDPβS blocks the signaling cascade, possibly by inhibiting the GDP–GTP exchange in Rho. As a result, in both cases, the transduction of the cascade remains blocked in the cells. Many GEFs are catalytically inactive when they form myosin complexes [77]. It has been described that blebbistatin can exert its inhibitory action on myosin functionality through the release of GEFs that stimulate the activation of Rac GTPase proteins in many cell types [77,78]. It is thus suggested that, in the presence of blebbistatin, the Rac- GTPase-protein-directed pathway should be active, which triggers the inhibition of the protein kinase that is responsible for the phosphorylation (and subsequent activation) of myosin [71,79]. Therefore, a study was conducted to determine whether GTP and its analogs caused the reversal of the inhibitory effect induced by blebbistatin. Because blebbistatin can induce disruption of actin–myosin interactions, the integrity of F-actin was assured in the presence of phalloidin. The addition of GTP to the blebbistatin-containing incubation medium caused even greater inhibition of teliospore motility than the drug alone, probably by stimulating the pathway that led to myosin dephosphorylation. How- ever, the presence of GDPβS, GTPγS, and above all, the joint addition of GTPγS and GTP succeeded in reversing the inhibitory effect of the drug. The GDPβS blocked the route that inhibits the functionality of myosin, so it must be active in the presence of the analog [66]. The recovery of mobility in the presence of GTPγS could again be the consequence of the inactivation of the route by hyperactivation of the Rac GTPase protein, which would avoid the blocking of the kinase that phosphorylates (and activates) myosin. Since the actions of the Rho and Rac GTPases are usually antagonistic [71], the inactivation of Rac would in turn lead to the activation of the Rho-triggered pathway (Figure9). Therefore, cell displacement is further stimulated during simultaneous incubation in the presence of GTPγS and GTP. During cell migration, the formation of protrusions as a result of the reorganization of actin filaments via the action of myosin II has been described in different eukaryotic cell types, including Dictyostelium, leukocytes, or fibroblasts [80,81]. The formation of filopodia and lamellipodia in all these cell types implies the translocation of the filaments toward the back of the protrusion as a result of the retrograde flow of the actin to create free space in the forward pole that allows for its polymerization in the positive (+) end of the filament [82]. Based on what occurs during cell displacement in other organisms, a movement model based on the reorganization of F-actin, in collaboration with myosin II (no protrusion formation), has been described for the migration of S. scitamineum cells [66] and is schematized in Figure9. First, the translocation movement of the actin filaments toward their negative end would produce the invagination of one of the cell poles. In turn, the translocation must be originated by the pushing movement of myosin II, which displaces the microfilaments toward the equatorial axis of the cell (negative (−) end of the filament). As a sine qua non condition, the actin filaments must be attached to the plasma membrane at one of the cell poles using membrane anchor proteins. In this way, when the filaments are “pushed” into J. Fungi 2021, 7, x FOR PEER REVIEW 12 of 19

The organization of the cytoskeleton in S. scitamineum cells is also a consequence of a signaling cascade triggered by the action of GTPase proteins [66]. The addition of GTP to the teliospores’ incubation medium partially reverses the inhibitory effect of latrunculin A, probably through Rho activation; in contrast, neither the addition of GTPγS, a non-hydrolyzable analog of GTP that keeps GTPase activated [74], nor the addition of GDPβS, a deactivator of Rho GTPases [75], were able to reverse the inhibitory effect. These results indicate that GTPγS may behave as a strong Rho activator such that cells may respond to this hyperactivation by inactivating the pathway. This suggests that Rho proteins should play an important role as a communication axis in the production and modulation of cellular responses to different forms of stress [76]. On the other hand, GDPβS blocks the signaling cascade, possibly by inhibiting the GDP–GTP exchange in Rho. As a result, in both cases, the transduction of the cascade remains blocked in the cells. Many GEFs are catalytically inactive when they form myosin complexes [77]. It has been described that blebbistatin can exert its inhibitory action on myosin functionality through the release of GEFs that stimulate the activation of Rac GTPase proteins in many cell types [77,78]. It is thus suggested that, in the presence of blebbistatin, the Rac-GTPase- protein-directed pathway should be active, which triggers the inhibition of the protein kinase that is responsible for the phosphorylation (and subsequent activation) of myosin [71,79]. Therefore, a study was conducted to determine whether GTP and its analogs caused the reversal of the inhibitory effect induced by blebbistatin. Because blebbistatin can induce disruption of actin–myosin interactions, the integrity of F-actin was assured in the presence of phalloidin. The addition of GTP to the blebbistatin-containing incubation medium caused even greater inhibition of teliospore motility than the drug alone, probably by stimulating the pathway that led to myosin dephosphorylation. However, the pres- J. Fungi 2021, 7, 44 ence of GDPβS, GTPγS, and above all, the joint addition of GTPγS and GTP succeeded13 of in 18 reversing the inhibitory effect of the drug. The GDPβS blocked the route that inhibits the functionality of myosin, so it must be active in the presence of the analog [66]. The recovery of mobility in the presence of GTPγS could again be the consequence ofthe the cell, inactivation they “pull” of the plasmaroute by membrane, hyperactiv producingation of the invagination Rac GTPase atprotein, one of which the poles would [83]. avoidOnce the the blocking invagination of the is kinase produced, that thephosph sporeorylates must move(and activates) forward (Figuremyosin.9 ).Since As the actionspolymerization of the Rho is notand interrupted, Rac GTPases the are positive usually (+) antagonistic end located [71], at the the non-invaginated inactivation of Rac end wouldgrows in rapidly; turn lead therefore, to the itactivation ends up “pushing”of the Rho-triggered the cell membrane, pathway undoing (Figure the 9). invagination Therefore, cellproduced displacement at the back is further end, favoringstimulated the during advance simultaneous of the teliospore, incubation and allowing in the presence the cycle of to GTPbeginγS again.and GTP.

Rho GTPase

Kinase GEF-

Ble

GEF Rac GTPase

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FigureFigure 9. 9. DiagramDiagram representing representing how how the the presence presence of of bleb blebbistatinbistatin (Ble) (Ble) could could trigger trigger the the activation activation ofof the the Rac Rac GTPase GTPase by by means means of of the the release release of oftheir their guanosine guanosine nucleotide nucleotide exchange exchange factors factors (GEFs). (GEFs). The activityThe of the activity Rac GTPase of the Rac would GTPase block would myosin block phosphorylation myosin phosphorylation via the corresponding via the corresponding ki- kinase. nase. In turn,In turn,the Rac the GTPase Rac GTPase inhibition inhibition would would stimulate stimulate Rho RhoGTPase GTPase activation activation and andpolymeriza- polymerization of the

tion of the actin.actin. :: myosin myosin II, II, : actin filaments. ﬁlaments.

During cellOn migration, the other the hand, formation MTs are of alsoprotrusions represented as a result in the of scheme the reorganization of Figure 10 showing of actin filamentswhy they via participate the action in of teliospore myosin II displacement. has been described It wasobserved in different that eukaryotic MTs were involved cell types,in including the motility Dictyostelium of teliospores, leukocytes, since nocodazoleor fibroblasts (Noc) [80,81]. decreased The formation its displacement of fi- to a maximum concentration of 0.5 µg mL−1 [66]. Moreover, Figure 11 shows variations in lopodia and lamellipodia in all these cell types implies the translocation of the filaments the agglutination of the spores in the presence of Noc. The results are expressed as a toward the back of the protrusion as a result of the retrograde flow of the actin to create percentage of cells involved in each of the different groups. It can be observed that the free space in the forward pole that allows for its polymerization in the positive (+) end of size of the cytoagglutinations formed in the presence of the drug decreased, probably as a the filament [82]. Based on what occurs during cell displacement in other organisms, a result of the inhibitory effect of Noc on chemotaxis [66]. Specifically, in the presence of Noc, movement model based on the reorganization of F-actin, in collaboration with myosin II it was observed that a greater number of cells involved in the medium-sized groups (51 to (no protrusion formation), has been described for the migration of S. scitamineum cells [66] 200 spores), which was accompanied by the absence of large clusters (groups of 200 to and is schematized in Figure 9. 500 teliospores). First, the translocation movement of the actin filaments toward their negative end On the other hand, it was proven that the dynamic interactions between the MTs would produce the invagination of one of the cell poles. In turn, the translocation must be and actin filaments direct the migration process in fibroblasts, where the complete de- originated by the pushing movement of myosin II, which displaces the microfilaments originatedpolymerization by the pushing of movement the MTs totally of myosin blocks II, its which displacement displaces [ 84the]. microfilaments The interaction between toward the equatorial axis of the cell (negative (−) end of the filament). As a sine qua non toward theMTs equatorial and actin axis filaments of the cell is (negative a basic phenomenon (−) end of the that filament). underlies As a all sine cellular qua non processes in condition, the actin filaments must be attached to the plasma membrane at one of the cell condition,which the actin it is filaments necessary must to establish be attached and to maintain the plasma cellular membrane asymmetry at one [85 of], the with cell these being poles usingsome membrane of the few anchor cellular proteins. types In in this which way, it when has been the filaments found that are the “pushed” cellular into movement is the cell, theytotally “pull” independent the plasma of membrane, the MTs [84 producing]. The interaction invagination of actin at andone MTsof the in polesS. scitamineum [83]. Oncecells the invagination was found. Labelling is produced, experiments the spore of must F-actin move with forward phalloidin (Figure conjugated 9). As the with Alexa polymerizationFluor ®is488 not showedinterrupted, differences the positive in the (+) organization end located ofat actinthe non-invaginated filaments in the end absence and grows rapidly;in the therefore, presence it of ends Noc up at “pushi a concentrationng” the cell of membrane, 0.5 µg mL− undoing1. The decrease the invagina- in the number tion producedof cells at showingthe back end, an asymmetric favoring th arrangemente advance of ofthe F-actin teliospore, inside and the allowing cell in the the presence of cycle to beginthe inhibitoragain. confirmed an interaction between the microfilaments and MTs during the On theestablishment other hand, MTs of cell are polarity also represente in S. scitamineumd in the scheme[50]. of Figure 10 showing why they participate in teliospore displacement. It was observed that MTs were involved in the motility of teliospores since nocodazole (Noc) decreased its displacement to a maximum concentration of 0.5 µg mL−1 [66]. Moreover, Figure 11 shows variations in the agglutination of the spores in the presence of Noc. The results are expressed as a percentage of cells involved in each of the different groups. It can be observed that the size of the cytoagglutinations formed in the presence of the drug decreased, probably as a result of the inhibitory effect of Noc on chemotaxis [66]. Specifically, in the presence of Noc, it was observed that a greater number of cells involved in the medium-sized groups (51 to 200 spores), which was accompanied by the absence of large clusters (groups of 200 to 500 teliospores).

J. FungiJ. Fungi 20212021, 7, ,7 ,x 44 FOR PEER REVIEW 14 of 18 14 of 19

3 3

2 2

1 1

FigureFigure 10. 10.Schematic Schematic representation representation (on (on the right)the right) superimposed superimposed on a real on imagea real (onimage the (on left) the that left) that demonstratesdemonstrates the the role role of theof the cytoskeleton cytoskeleton in teliospore in teliospore motility. motili Thety. binding The binding of the quorum of the quorum signal signal toto its its receptors receptors (1) (1) induces induces polar polar cell cell invagination invagination (2), (2) which, which is produced is produced by the by interaction the interaction of an of an ATPaseATPase with with contractile contractile ability, ability, which which is sensitive is sensitive to blebbistatin, to blebbistatin, with F-actin with cytoskeleton. F-actin cytoskeleton. After this, After thethis, depolymerization the depolymerization of F-actin of F is-actin achieved is achieved at the opposite at the opposite pole, the pole, repolymerization the repolymerization of which of which producesproduces the the cell cell advancement advancement (3). (3). Throughout Throughout thisprocess, this process microtubules, microtubules (MTs) are (MTs reorganized) are reorganize in d in cellscells to to collaborate collaborate in in the the displacement displacement with with F-actin F-actin ﬁlaments. filaments. Scalebars Scale indicate bars indicate 2 µm. 2 µm.

ControlOn the cells, other which hand, were it was not proven treated withthat anythe dynamic of the described interactions inhibitors, between revealed the a MTs and homogeneousactin filaments distribution direct the ofmigration actin ﬁlaments process by in the fibroblasts, cytoplasm where before the cell complete polarization, depolymer- while the MT showed a directed polymerization (polarized) toward a single point of the ization of the MTs totally blocks its displacement [84]. The interaction between MTs and cell, probably the one corresponding to the germinative pole, which was co-indicated with theactin actin filaments capping whenis a basic the cell pheno polarizedmenon [50 that]. underlies all cellular processes in which it is necessaryFinally, to although establish cytoskeleton and maintain organization cellular asymmetry during chemotaxis [85], with and these germination being some is of the essentialfew cellular during typesS. scitamineum’s in which it hasearly been pathogenicity, found that it the is importantcellular movement to point out is thattotally inde- MTspendent could of be the involved MTs [84]. in the The conjugation interaction process of actin by and means MT ofs in their S. scitamineum interaction with cells the was found. actinLabelling cytoskeleton experiments [52]. In addition,of F-actin MTs with are traditionally phalloidin required conjugated to generate with cytokineticAlexa Fluor® 488 phragmoplastshowed differences during cellin the division organization in plants [of86 ];actin therefore, filaments polymerization in the absence could and also in be the pres- involvedence of Noc in hyphae at a concentration septation, which of would0.5 µg explainmL−1. The why decrease germinative in the tubes number (even if of empty) cells showing are not released to the media in the presence of nocodazole [50]. an asymmetric arrangement of F-actin inside the cell in the presence of the inhibitor confirmed an interaction between the microfilaments and MTs during the establishment of cell polarity in S. scitamineum [50].

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Figure 11. Cytoagglutination degree (number of teliospores per aggregate and number of total aggregates in the media) Figure 11. Cytoagglutination degree (number of teliospores per aggregate and number of total aggregates in the media) that that was induced by distilled water (black) or 0.5 µg mL−1 nocodazol (gray). Values are the mean of three replicates. Ver- was induced by distilled water (black) or 0.5 µg mL−1 nocodazol (gray). Values are the mean of three replicates. Vertical bars tical bars give the standard errors, where larger than the line (A). Teliospores aggregate formation induced by water (B) give the standard errors, where larger than the line (A). Teliospores aggregate formation induced by water (B) or nocodazol or nocodazol (C). Scale bars indicate 50 µm. (C). Scale bars indicate 50 µm. Control cells, which were not treated with any of the described inhibitors, revealed a 8. Conclusions homogeneous distribution of actin filaments by the cytoplasm before cell polarization, while theIt can MT be showed concluded a directed that some polymerization cellular processes (polarized) are essential toward in aS. single scitamineum point ofduring the cell,sugar probably cane infection. the one corresponding First, arginase isto synthesizedthe germinative by teliospores pole, which of wasSporisorium co-indicated scitamineum with thewhen actin environmental capping when conditionsthe cell polarized are adequate [50]. for their proliferation. Synthesized and secretedFinally, arginase although induces cytoskeleton a QS effect organizati that promoteson during infection chemotaxis since it and agglutinates germination enough is teliospores. Moreover, the arginase produced in the early stages stimulates germination essential during S. scitamineum’s early pathogenicity, it is important to point out that MTs of the cytoagglutinated teliospores that were “ready for the attack.” On the other hand, could be involved in the conjugation process by means of their interaction with the actin the formation of protrusions that were derived from cytoskeleton interactions is indis- cytoskeleton [52]. In addition, MTs are traditionally required to generate cytokinetic pensable for cell migration, namely, with actin, myosin, and MTs, as well as the signaling phragmoplast during cell division in plants [86]; therefore, polymerization could also be cascade mediated by the GTPases that collaborate in both germination and displacement involved in hyphae septation, which would explain why germinative tubes (even if Finally, it is easy to understand why plant resistance mechanisms are focused on empty) are not released to the media in the presence of nocodazole [50]. trying to reorganize the teliospore cytoskeleton “at its whim.” This is why chemoattractive proteins trigger cytoskeleton organization to collect teliospores to a cytoagglutination 8. Conclusions point. However, at the same time, sugar cane arginase causes cytoskeleton disorganization, whereIt can germination be concluded does that not some take cellular place and processes infection are does essential not progress. in S. scitamineum during sugar cane infection. First, arginase is synthesized by teliospores of Sporisorium scitami- neumAuthor when Contributions: environmentalAll authors conditions participated are adequa in the datate collectionfor their andproliferation. article design, Synthesized including the andarticle secreted writing, arginase chapter design, induces and a graphic QS effect design. that All promotes authors have infection read and since agreed it toagglutinates the published enoughversion teliospores. of the manuscript. Moreover, the arginase produced in the early stages stimulates ger- minationFunding: ofThis the researchcytoagglutinated was supported teliospores by funds that from were project “ready UCM, for the GR3/14.910081 attack.” On the from other Com- hand,plutense the Universityformation (Spain). of protrusions that were derived from cytoskeleton interactions is indispensable for cell migration, namely, with actin, myosin, and MTs, as well as the sig- Conﬂicts of Interest: The authors declare no competing ﬁnancial interest. naling cascade mediated by the GTPases that collaborate in both germination and displacement Finally, it is easy to understand why plant resistance mechanisms are focused on trying to reorganize the teliospore cytoskeleton “at its whim.” This is why chemoattractive

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