Polyamines levels increase in smut teliospores after contact with sugarcane glycoproteins as a plant defensive mechanism
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Previous studies have already highlighted the correlation between Sporisorium scitamineum pathogenicity and sugarcane polyamine accumulation. It was shown that high infectivity correlates with an increase in the amount of spermidine, spermine and cadaverine conjugated to phenols in the sensitive cultivars whereas resistant plants mainly produce free putrescine. However, these previous studies did not clarify the role of these polyamides in the disorders caused to the plant. Therefore, the purpose of this research is to clarify the effect of polyamines on the development of smut disease. In this paper, commercial polyamines were firstly assayed on smut teliospores germination. Secondly, effects were correlated to changes in endogenous polyamines after contact with defense sugarcane glycoproteins. Low concentrations of spermidine significantly activated teliospore germination, while putrescine had no activating effect on germination. Interestingly, it was observed that the diamine caused nuclear decondensation and breakage of the teliospore cell wall whereas the treatment of teliospores with spermidine did not induce nuclear decondensation or cell wall breakdown. Moreover, the number of polymerized microtubules increased in the presence of 7.5 mM spermidine but it decreased with putrescine which indicates that polyamines effects on Sporisorium scitamineum teliospore germination could be mediated through microtubules interaction. An increased production of polyamines in smut teliospores has been related to sugarcane resistance to the disease. Teliospores incubation with high molecular mass glycoproteins (HMMG) from the uninoculated resistant variety of sugarcane, Mayari 55-14, caused an increase of the insoluble fraction of putrescine, spermidine and spermine inside the teliospore cells. Moreover, the level of the soluble fraction of spermidine (S fraction) increased inside teliospores and the excess was released to the medium. The HMMG glycoproteins purified from Mayarí 55-14 plants previously inoculated with the pathogen significantly increased the levels of both retained and secreted soluble putrescine and spermidine. Polyamines levels did not increase in teliospores after incubation with HMMG produced by non resistant variety Barbados 42231 which could be related to the incapacity of these plants to defend themselves against smut disease. Thus, a hypothesis about the role of polyamines in sugarcane-smut interaction is explained.
KeywordsMicrotubules Putrescine Smut Spermidine Spermine Sugarcane Teliospores
- B 42231
Barbados 42231 cv.
Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid
High molecular mass glycoproteins
High Molarity Pipes Buffer
Mid molecular mass glycoproteins
- My 55-14
Mayarí 55-14 cv.
Phosphate saline buffer
Insoluble conjugated polyamine fraction
Soluble, free polyamine fraction
Soluble conjugated polyamine fraction
Smut, caused by the fungus Sporisorium scitamineum (Syd.), is one of the major diseases of sugarcane (Saccharum officinarum L.). It causes severe epidemic episodes in growing areas around the world and affects the vegetative development and juice quality of plants, reducing crop yields by 20–30%, with huge economic impact for losses that entails (Que et al. 2014).
The infection process was already described by Waller in 1970. Firstly, smut teliospores come in contact with the buds of the plant. Then, fungal cells germinate and release haploid sporidia that conjugate to form an infective fungal mycelium (Trione 1990). Polarity of the cell is absolutely required for the onset of germination (Fontaniella et al. 2002). In particular, organized microtubules (MT) are responsible for condensation, correct organization and migration of the nucleus through the hypha (Sanchez-Elordi et al. 2016a), which penetrates the stem and reaches the meristematic region, inducing the formation of a modified reproductive structure known as a whip (Carvalho et al. 2016). Smut sporogenesis has been described in detail by Marques et al. (2017). These authors have demonstrated that the spores are exclusively formed at the base of the whip. In this structure, teliospore black masses accumulate to be then released and scattered to other stems by the wind to start a new infection cycle.
Defense of sugarcane plants depends on a multifactorial response. De Armas et al. (2007) have correlated the susceptibility or resistance to smut to changes in free phenolic compound levels and phenylalanine ammonia lyase (PAL) and peroxidase activities. It has been recently demonstrated that resistant sugarcane varieties respond to the infection by the formation of a protective barrier layer with lignin, cellulose and arabinoxylan in the cell walls (Marques et al. 2018). On the other hand, innovating comparative genomic studies are focused on the analysis of highlighting genes with a potential role in host-pathogen interaction (Benevenuto et al. 2018) as it had been described in relation to expression levels of dirigent proteins transcripts in sugarcane (Sánchez-Elordi et al. 2018).
Sugarcane produces mid molecular and high molecular mass glycoproteins (MMMG and HMMG) in response to pathogenic infections, wounds or several types of stress (Legaz et al. 1995). A fraction of active HMMG glycoproteins isolated from a resistant to smut sugarcane cultivar (cv.) contains several polypeptides with different biological activities involved in the resistance (Legaz et al. 1995, 1998b; Millanes et al. 2005). Interestingly, arginase activity contained in HMMG from resistant plants seems to be responsible for preventing germination of agglutinated smut teliospores (Sánchez-Elordi et al. 2016b) by means of MT depolymerization and nuclear decondensation (Sánchez-Elordi et al. 2016a).
Arginase is traditionally linked to polyamines (PAs) biosynthesis. Many researchers have highlighted the obvious correlation between fungal pathogenicity and PAs accumulation (Rajam et al. 1985; West and Walters 1989; Walters 2003). In fungi, variations of the concentration of putrescine (PUT) and spermidine (SPD) are closely associated to the onset of important physiological processes for the development of the organism (Walters 1995). Thus, it has been shown that the addition of low concentrations (0.1–1 mM) of PUT and SPD to the culture media leads to the increased growth rate of some phytopathogenic fungi (Rajam and Galston 1985). SPD, in particular, seems to be the most prevalent fungal PA (Walters 1995). Although PUT does not seem to be an essential factor for cell growth, it is involved in the protection mechanisms against different forms of stress and in virulence of Ustilago maydis (Valdes-Santiago et al. 2010).
On the other hand, it is well established that PAs are essential for growth and development of plants. They are involved in important physiological process (Gill and Tuteja 2010) such as in tolerance against abiotic stress (Gupta et al. 2013; Gill and Tuteja 2010). However, the specific role of plant PAs in the biotic defense is not yet well known, although important studies are being carried out. Wojtasik et al. (2015) observed an increased expression of genes involved in PA biosynthesis of flax plants after infection by Fusarium, as well as the concomitant increase of the total amount of PAs in infected plant tissues. Furthermore, in vitro experiments demonstrated that PAs play an inhibitory role in Fusarium growth. PAs increase following infection could be related to the role of PAs in the activation of some enzymes involved in the degradation of pathogen cell walls (Charnay et al. 1992). Defensive role of PAs could also be a consequence of their binding to some molecules inside the cells, disabling important processes for the pathogen development.
The latter is possible because of the polycationic nature of PAs at physiological pH values. PAs are able to bind strongly to existing negative charges in different cellular components, such as nucleic acids, proteins and phospholipids (Gupta et al. 2013). For this reason, they are good candidates for binding to cytoskeletal proteins with negative charges, such as actin or tubulin, promoting their polymerization (Grant et al. 1983; Pohjanpelto et al. 1981). Specifically, it has been found that PAs significantly modulate MT assembly in eukaryotic cells (Savarin et al. 2010). This is because PAs favor the nucleation process and increase the attraction between tubulin heterodimers influenced by their C-terminal ends, which are highly negatively charged. In addition, PAs generate an attractive force that contributes to the meeting of GTP-tubulin units and the growing filament (Mechulam et al. 2009), enhancing its elongation.
Thus, we propose in this work that the role of arginase activity in sugarcane defense against smut could be related to a change in PAs levels in fungal cells that prevents germination by means of cytoskeleton blockade.
Materials and methods
Biological material and inoculation process
Teliospores of S. scitamineum collected from whips developed in diseased Barbados 42,231 (B 42,231) cv., field-grown in “Antonio Mesa” Sugarcane Experimental Station (Jovellanos, Matanzas, Cuba), and sugarcane plants, field grown in the Real Jardín Botánico Alfonso XIII (Madrid, Spain), were used throughout this work. Sugarcane Mayarí 55-14 (My 55-14) cv., resistant to smut, and B 42,231 cv., sensitive to smut, were employed. All the chemicals, except when indicated, were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
Inoculation of plants from resistant and susceptible varieties was conducted as it is described in Sánchez-Elordi et al. (2016a). Stems from inoculated and non-inoculated plants of My 55-14 and B 42,231 varieties were used to extract juice from which the defensive glycoproteins (High Molecular Mass Glycoproteins, HMMG) were obtained to be used in assays.
Purification of HMMG from total juice
HMMG sugarcane glycoproteins from inoculated and non-inoculated plants of My 55-14 and B 42231varieties were obtained from stalks as previously described (Sanchez-Elordi et al. 2015). Proteins concentration in HMMG (fraction used for experiments) was determined according to the method of Lowry et al. (1951).
Effect of polyamines on teliospore germination
Dry teliospores (1.0 mg) were pre-hydrated in distilled water for 1 h (1 mg mL− 1). To evaluate if PAs modify teliospore germination, aliquots of 100 µL of this suspension were placed in a Neubauer chamber to which 100 µL of PUT or SPD solutions were added to reach a final concentration ranging from 0.1 mM to 10 mM, and maintained at 25 °C for 15 h. A sample containing distilled water instead of PA solutions was included as control. Percent of germination in presence of the different PAs was evaluated by using a light Olympus BX51 microscope fitted with an Olympus DP72 camera for capturing images. Images were analyzed by using the Cell A Image Acquisition Software. Each analysis was performed in triplicate.
In vitro polymerization of tubulin isolated from smut teliospores
Partial purification of tubulin from smut teliospores was achieved according to that described by Castoldi and Popov (2003) with modifications. Then, polymerization assays were performed in spectrophotometric cuvettes containing 100 µL (0.11 mg) tubulin, 600 µL glycerol, 600 µL High Molarity Pipes Buffer (HMPB) and 200 µL of MilliQ water. HMPB, pH 6.9, was composed by 1M PIPES [piperazine-N, N’-bis (2-ethanesulfonic acid)], 10 mM MgCl2 and 20 mM EDTA. When indicated, PUT or SPD were added to the mixture to attach a final concentration of 0.5 and 7.5 mM PA. Finally, ATP and GTP were added in samples to trigger the polymerization (final concentration of 1.5 mM and 0.5 mM, respectively). Tubulin polymerization was followed by measuring the increase of absorbance at 350 nm at 37 °C in samples.
Polyamines impact on nuclear organization: DAPI staining
0.25 mg of dry teliospores were pre-hydrated in distilled water for 1 h (1 mg mL− 1). Then, 100 µL of this teliospore suspension were incubated with 400 µL of increasing concentrations of PUT or SPD (0–10 mM) for 15 h in shaking. A control without PAs was performed. After 15 h, PAs or water were discarded and 50 µL of formaldehyde was added to samples for fixation. The fixation solution was also discarded after 10 min and teliospores were washed with 100 µL water for 10 min. In order to facilitate the fluorescence entering, samples were incubated in 100 µL of 7% polyvinylpyrrolidone (PVP) for 10 min with vigorous shaking. Without removing the PVP, 60 µL of 20 µM of 4,6-diamidino-2-phenylindole, DAPI (Sigma-Aldrich, Saint Louis, MO, USA), were added for nuclear staining. Teliospores were incubated for 10 min with vigorous shaking and in the dark. Samples were washed with distilled water to eliminate the excess of fluorescence and mounted in Mowiol-DABCO (Calbiochem) to minimize fluorescence bleaching.
Light and fluorescence images were obtained using an Olympus BX60 fluorescence microscope fitted with an Olympus DP70 camera and analyzed by the use of a DP Controller 2002 (Olympus). The 405 nm line was used for excitation and emission was detected at 461 nm.
Extraction and analysis of polyamines
1.0 mg mL− 1 spore samples were placed in distilled water for 1 h. Then, the suspensions were centrifuged for 1 min at 9000×g. The supernatant was discarded, while the precipitate was incubated with 0.16 mg in 1.0 mL distilled water of HMMG fractions from inoculated and non-inoculated plants from Mayarí and Barbados varieties with continuous stirring for 3 h. After this, the samples were newly centrifuged for 1 min at 9000×g. The supernatant obtained contain PAs secreted by teliospores. PAs were extracted from teliospores or from the incubation media using the method described by Legaz et al. (1998b). Briefly, samples of 0.25 g of smut teliospores (1.0 mL of incubation medium) were macerated with 50 mg alumina and 3.0 mL 5% (v/v) perchloric acid. In addition, 2.0 mL of 5% (v/v) perchloric acid was added to 750 µL of the incubation media. These suspensions were stored overnight at 4 °C in plastic tubes and then centrifuged at 48 000×g for 20 min at 2 °C. Pellets obtained after this centrifugation contained PH fraction. The supernatants, containing free PAs and acid-soluble PAs (S + SH fraction), were dried at 70 °C under a stream of air and resuspended in 2.0 mL of 2 × distilled water. Perchloric acid from supernatants was extracted with 3 × 5 ml of ethyl ether. After separation by centrifugation at 5000×g, ether containing perchloric acid was discarded. PH and S + SH fractions were hydrolyzed with 12 N HCl at room temperature for 18 h. After hydrolysis, samples were dried in air flow and dry residues were resuspended in 2.0 mL of 5% (v/v) perchloric acid and centrifuged at 17,600×g 20 min at 2 °C. Supernatants contained soluble PAs from S, SH and PH fractions.
Separation and quantitation of polyamines by capillary electrophoresis
PA content of the different fractions was analyzed by capillary electrophoresis using a CE system P/ACETM MDQ Glycoprotein from Beckman Coulter (Fullerton, CA, USA). The system incorporated a Diode Array detector with a bandwidth of 6 nm. Electropherograms were obtained by a computer coupled to the system, using the program 32 KaratTM. The analytical conditions were those described by Arce et al. (1997), with modifications: migration buffer, 4.0 mM CuSO4, 4.0 mM formic acid and 4.0 mM 18-ether crown/6, pH 3.0; hydrodynamic injection at 0.5 psi for 7.0 s; capillary temperature, 20 °C; sample temperature, 15 °C; constant voltage at 12 kV with anode-to-cathode polarity; indirect detection at 210 nm.
Spermidine and putrescine have opposite effects on smut teliospore germination
To address the possible role of PAs on teliospore germination, we seeded teliospores in germinative medium in the presence of different concentrations of PUT or SPD. We found that PUT had no activating effect on germination (Fig. 1a, e). The germination percentage in the presence of PUT remains below that of control conditions (C) throughout the experiment, and there is a dose-dependent inhibition of germination, which is most apparent at the highest concentration used (10 mM). In contrast, a supply of 0.1 mM SPD to the incubation media of teliospores activated germination from 20 h incubation (Fig. 1b, d). Higher concentrations did not cause this effect. 7.5 mM SPD decreased germination. 10 mM SPD caused 100% inhibition.
Spermidine promotes in vitro polymerization of semi-purified tubulin S. scitamineum, whereas putrescine inhibits it
Due to their effect on the emergence of the germinative tube, we hypothesized that PAs had a differential effect on MT polymerization. To address this, we performed in vitro MT polymerization in the presence of SPD. Using semi-purified smut tubulin, we observed that SPD altered polymerization dynamics (results shown in Fig. 2). MT polymerization in the presence of 7.5 mM SPD was increased as early as 15 min after initiation of the experiment, reaching a 2.5 fold induction after 150 min. Low concentrations had no detectable effect. Conversely, PUT decreased MT polymerization, which was maximal at 7.5 mM.
Putrescine triggers DNA decondensation and cell wall breakage
To address the cellular effects of PUT and SPD, we examined teliospores incubated with either PA by light microscopy using DAPI to stain nuclear DNA. We found that PUT caused nuclear decondensation and breakage of the teliospore cell wall. Cell wall breakdown (Fig. 3c) was related to the appearance of sterile fungal cell walls (Fig. 3g) and to the appearance of protoplasts (Fig. 3c, e) defined as the cell content expelled outside the teliospore upon breakdown of the cell wall. Note the decondensed nuclei in Fig. 3f. Conversely, treatment of teliospores with SPD caused no apparent damage. We did not observe nuclear decondensation or cell wall breakdown (Fig. 4a–f). Instead, we noticed larger numbers of germinated spores and sporidia release even at the lowest concentration of SPD (Fig. 4c, e). Only 10 mM SPD caused nuclear decondensation (Fig. 4g, h).
Quantitation of polyamine content and secretion from teliospores of S. scitamineum by capillary electrophoresis
The results of analysis of PAs by capillary electrophoresis can be observed in Fig. 5. PAs levels in samples were quantified by capillary electrophoresis from the times of migration of the three PAs valuated (Fig. 6b). Increasing concentrations of the patters were used to elaborate calibration curves (Fig. 6d) applicable to the teliospores samples (Fig. 6c).
Results for free acid-soluble and conjugated acid-insoluble PAs in secreted and internal fractions of fungal cells are shown. In any case conjugated soluble PAs were found. Moreover, results confirmed that HMMG fractions from different juices had not detectable PAs before contact with teliospores (results not shown).
The spores which have not been pre-incubated with the HMMG fraction (control sample) contained both PUT and SPD in soluble form (S fraction). Levels of both PAs were kept below 0.3 mg mg dry weight teliospore− 1. In addition, both PAs were secreted (around 0.1 m mg teliospore− 1).
Incubation of teliospores with HMMG caused changes in the production and secretion of PAs with respect to the control. Teliospores incubation on HMMG fraction from the non-inoculated resistant variety, My 55-14, caused a rise of PUT, SPD and SPM insoluble fractions inside the cells. Moreover, the level of the secreted and accumulated acid-soluble SPD significantly increased in relation to the control. On the other hand, incubation of teliospores in the presence of HMMG from inoculated My 55-14 plants significantly increased the levels of both retained and secreted soluble PUT. Secretion of acid-soluble SPD and acid-soluble SPM are also observed. It might be noted that SPM production was only detected after incubation with My 54-14 glycoproteins.
Regarding the incubation of smut teliospores in the presence of HMMG from the sensitive variety, B 42,231, results showed a decrease of the levels of soluble PUT and a slight increase of the levels of SPD inside the cells of about 0.25 mg PA per mg spore. These results have been summarized in Fig. 6.
The main pathway of PA synthesis in fungi requires an ornithine decarboxylase (ODH) to synthesize PUT (Valdés-Santiago et al. 2012). This diamine derives from arginine, which is hydrolyzed by arginase to ornithine, the substrate of ODH and urea (Walters 1995).
This report provides novel evidence on the role of PAs in the infectivity of smut and in the defense of sugarcane plants. We have previously shown that resistant cvs of sugarcane produce levels of arginase higher than those produced by sensitive varieties when invaded by the pathogen (Sánchez-Elordi et al. 2016a). Such activity would trigger ornithine conversion into PUT by ODH. This would be in agreement with the results shown in Figs. 5 and 6, in which the resistant cv., My 55-14, almost doubles the level of total PAs after infection against an uninfected control. The sensitive cv., B 42,231 however, does not modify its level of total PAs after infection. Regarding the potential infectivity of smut, we also showed that low concentrations of PUT promoted the appearance of sporidia, which would strengthen the infective potential of the pathogen (Sánchez-Elordi et al. 2015). Our results demonstrate that germination decreased after incubation of the spores with high concentrations of exogenous PUT or SPD. Therefore, the induction of germination and infective capacity of the pathogen displays a dose-dependent relationship with the concentration of PAs, either exogenous or produced by the teliospores themselves in an uncontrolled manner.
The most common mode of action of Ustilago maydis is similar to that of S. scitamineum, causing the smut of corn and sugarcane, respectively. U. maydis promotes a saprophytic phase, non-infective, growing as a budding yeast. In a second phase, a highly infective dikaryotic mycelium is produced by the mating of two sexually compatible yeast-like cells. Teliospores of S. scitamineum germinate to produce oval, haploid cells called sporidia, which fuse when they are sexually compatible to form a diploid infective mycelium. Then, an apressorium-like structure seems to be responsible for penetration in plant tissues (Marques et al. 2018). PUT is important for the transformation into infective mycelium as U. maydis unable to produce PUT was also unable to achieve the transition to the dimorphic state (Guevara-Olvera et al. 1997), although growth was not inhibited. However, it was able to grow and to produce a dimorphic form after exogenous supplementation with SPD (Valdés-Santiago et al. 2010). Together with the results shown in Fig. 1, this would imply that teliospore germination requires a very efficient and rapid conversion of PUT into SPD. If, on the other hand, PUT is allowed to accumulate, germination would fail and no compatible sporidia would appear.
In addition, PUT damages the cell wall, promotes DNA decondensation and it also prevents MT polymerization (Fig. 2), which is consistent with the observed increase in the insoluble fraction of the PA content after treatment with HMMG from non-inoculated My 55-14. However, the precise biological mechanisms by which PAs affect these processes remains uncertain. PUT induces apoptosis in mammalian cells (Takao et al. 2006), which may be related to nuclear decondensation. A similar mechanism could promote spore death and DNA decondensation, which is a hallmark of apoptosis, leading to global repression of the pathogen. Furthermore, PUT promotes the silencing of survival-related genes in pathogenic fungi, such as Aspergillus nidulans (Khatri and Rajam 2007) and phytopathogens, such as Magnaporthe oryzae (Kadotani et al. 2003).
On the other hand, a low concentration of SPD triggers germination. Although this low concentration does not affect MT polymerization in vitro, higher concentrations enhance it. It is likely that high concentrations may lead to excessive MT polymerization, which would be detrimental for germination and/or growth.
According to our model of PA production by infected sugarcane tissues as a defense mechanism against the pathogen (Legaz et al. 1998a; Piñón et al. 1999), a short-term action, as described in Fig. 1, would require an effector PUT production formed as a response to the infection. This effector cannot be salicylate, since the increase caused by this acid on the production of HMMG occurs for time periods longer than 7 days (Fontaniella et al. 2002). There may be a very short-term response regulated, perhaps, by the production of jasmonic acid, which may induce arginase (Brauc et al. 2012).
The incubation of spores with HMMG obtained from experimentally infected, resistant cv. leads to a remarkable increase of the concentration of both soluble and small molecule-conjugated PAs inside teliospores, which is in agreement with previous results of our group regarding PA conjugation to sugarcane phenols after experimental plant infection (Legaz et al. 1998a). The most interesting thing is the fact that HMMG from the susceptible variety (before or after inoculation) do not increase PA levels in fungal cells, which must be necessarily related to the defense incapacity of susceptible plants.
PAs conjugated to macromolecules were only detected when the teliospores were incubated in the presence of HMMG from non-inoculated plants (Fig. 6). It is proposed that conjugation of PAs to positively charged macromolecules (e.g. cytoskeletal components) could block germination of teliospores (Fig. 1). None of the conjugated PAs were secreted by the teliospores to the incubation medium, although they were secreted as soluble forms, mainly SPD and SPM, after incubation on HMMG from resistant cv. It is noteworthy that S. scitamineum is able to synthesize SPM, whereas U. maydis is not (Valdés-Santiago et al. 2009).
Finally, a hypothetical scheme of PAs role in the first stages of sugarcane-S. scitamineum interaction is proposed (Figs. 7, 8). Active teliospores produce their own arginase (Sánchez-Elordi et al. 2015) that binds to their receptors on the cell wall, inducing cytoagglutination and quorum acquisition to permit entry into the plant tissues. On the other hand, a part of the fungal arginase would be internalized increasing then the amount of endogenous PUT, which would be rapidly and efficiently converted into SPD. This SPD appears to induce polymerization of the tubulin while the excess of PUT would be secreted, favoring then the release of sporidia (Sánchez-Elordi et al. 2016a) (Fig. 7). However, the effect of HMMG from My 55-14 includes the blockage of the teliospore wall receptors by the plant arginase triggering a process of cytoagglutination and false quorum as it was previously described (Sánchez-Elordi et al. 2015). Then an uncontrolled excess of cane arginase would be internalized by teliospores, greatly increasing the amount of endogenous PUT, which induces nuclear decondensation, inhibition of germination and release of protoplasts (Fig. 8). The excess PAs would be conjugated to macromolecules or secreted to the medium.
It is suggested in this work that PAs at moderate levels are necessary to growth and pathogenicity of S. scitamineum. However, a deregulation of PAs levels causes injurious effect in cells. That is taken in advantage by sugarcane glycoproteins which induce an uncontrolled increase of PAs that leads to nuclear decondensation, cell wall breakdown and germinative blockage of teliospores as a part of plant defense mechanism.
- Carvalho G, Quecine MC, Longatto DP, Peters LP, Almeida JR, Shyton TG, Silva MML, Crestana GS, Crete S, Monteiro-Vitorello CB (2016) Sporisorium scitamineum colonisation of sugarcane genotypes susceptible and resistant to smut revealed by GFP-tagged strains. Annal Appl Biol 169:329–341CrossRefGoogle Scholar
- Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275Google Scholar
- Marques JPR, Appezzato-da-Glória B, Pipebring M, Massola N, Monteiro-Vitorello CB, Vieira MLC (2017) Sugarcane smut: shedding light on the development of the whip shaped sorus. Ann Bot 11:815–827Google Scholar
- Sánchez-Elordi E, Contreras R, de Armas R, Benito MC, Alarcón B, de Oliveira E, del Mazo C, Díaz-Peña EM, Santiago R, Vicente C, Legaz ME (2108) Differential expression of SofDIR16 and SofCAD genes in smut resistant and susceptible sugarcane cultivars in response to Sporisorium scitamineum. J Plant Physiol 226:103–113CrossRefGoogle Scholar