Melanin,DNA replication,and autophagy affect appressorium development in Setosphaeria turcica by regulating glycerol accumulation and metabolism

GUO Xiao-yue,LlU Ning,LlU Bing-hui,ZHOU Li-hong,CAO Zhi-yan,HAN Jian-min,DONG Jin-gao

State Key Laboratory of North China Crop Improvement and Regulation/Key Laboratory of Hebei Province for Plant Physiology and Molecular Pathology/Mycotoxin and Molecular Plant Pathology Laboratory,Hebei Agricultural University,Baoding 071001,P.R.China

Abstract Setosphaeria turcica (syn.Exserohilum turcicum) is the pathogenic fungus of maize (Zea mays) that causes northern leaf blight,which is a major maize disease worldwide. Melanized appressoria are highly specialized infection structures formed by germinated conidia of S.turcica that infect maize leaves. The appressorium penetrates the plant cuticle by generating turgor,and glycerol is known to be the main source of the turgor. Here,the infection position penetrated by the appressorium on maize leaves was investigated,most of the germinated conidia entered the leaf interior by directly penetrating the epidermal cells,and the appressorium structure was necessary for the infection,whether it occurred through epidermal cells or stomata. Then,to investigate the effects of key factors in the development of the appressorium,we studied the effects of three inhibitors,including a melanin inhibitor (tricyclazole,TCZ),a DNA replication inhibitor (hydroxyurea,HU),and an autophagy inhibitor (3-methyladenine,3-MA),on appressorium turgor and glycerol content. As results,appressorium turgor pressure and glycerol concentration in the appressorium reached their highest levels at the mature stage of the appressorium under the control and inhibitor treatments.The three inhibitors had the greatest effects on appressorium turgor pressure at this stage. Glycogen and liposomes are the main substances producing glycerol. It was also found inhibitors affected the distribution of glycogen and liposomes,which were detected in the conidia,the germ tube,and the appressorium during appressorium development. This study provides profound insight into the relationship between appressorium turgor pressure and glycerol content,which was affected by the synthesis of melanin,DNA replication,and autophagy in the developing appressorium during a S.turcica infection.

Keywords:melanin,DNA replication,autophagy,appressorium,development,Setosphaeria turcica, glycerol

1.lntroduction

Northern maize leaf blight is caused by the fungal pathogenSetosphaeriaturcicaresulting in devastating yield losses of maize (Shenet al.2013). The appressorium is a specialized infection structure produced by hyphae and the conidium,which is utilized to invade the cuticle,and colonize host tissues during the invasion of maize leaves byS.turcica(Zhanget al.2012;Guet al.2014). Therefore,complete development of the appressorium is necessary for successful invasion of maize leaves byS.turcica. However,the mechanism of appressorium development during infection is not fully understood.

The mature appressorium forms at the tip of the germ tube inMagnaporthe oryzaeandCollectotrichumspecies,and the accumulation of 1,8-dihydroxynaphthalene(DHN) melanin in the appressorium plays an important role in fungal penetration into the epidermis of the host(Gessleret al.2014;Ryder and Talbot 2015). DHN melanin accumulates in the appressorium cell wall where it changes the porosity of the cell wall. Thus,the efflux of molecules in the cell is hindered and intracellular glycerol concentration increases. The osmotic potential increases,causing influx of water into the cell and high turgor pressure. It has been reported that turgor in theM.oryzaeappressorium is >8.0 MPa,which enables the appressorium to penetrate the hard epidermis of rice (Howardet al.1991).Setosphaeria turcicaconidia contact the maize leaves and germinate under suitable conditions. Germination proceeds through the extension of a narrow germ tube that emerges from the conidia within 6 h of contact with the leaf surface (Caoet al.2007).The germ tube starts to swell at its apex within 10 h and flattens against the surface of the maize leaf;the germ tube apex develops into the appressorium. The mature appressorium produces invasive hyphae and adheres tightly to the leaf surface. InS.turcica,DHN melanin accumulates between the cell wall and membrane(Butler and Day 1998;Hensonet al.1999;Nosanchuk and Casadevall 2006;Caoet al.2011). These invasive hyphae grow into secondary hyphae in the host after penetrating the cuticle. TheS.turcicaappressorium is strongly melanized,and non-melanized appressoria fail to generate sufficient turgor and lose pathogenicity (Caoet al.2011).

The formation of an early appressorium is blocked inM.oryzaewhen the germinated conidia are treated with the DNA replication inhibitor HU. In addition,the infection nail and infection are inhibited when a mature appressorium is treated with HU (Saunderset al.2010;Osés-Ruizet al.2017). Plant infection byM.oryzaerequires the S-phase cell-cycle checkpoint that acts through the DNA damage response (DDR) pathway involving Cds1 kinase (Osés-Ruizet al.2017). Eukaryotes activate an important and conservative monitoring mechanism called the S-phase checkpoint to preserve genomic integrity and cellular viability (Zenget al.2020).

Autophagy is a ubiquitous and evolutionarily conserved process in which long-lived proteins and organelles are degraded in all eukaryotic cells (Levine and Klionsky 2004). Beginning with the identification of autophagyrelated (ATG) genes in yeast,studies of homologues in higher eukaryotes have reported that autophagy involves many aspects of physiology (Klionsky 2010). Autophagy is an important pathway of turgor accumulation in the appressorium (Veneault-Fourreyet al.2006;Liuet al.2007). TheM.oryzaeappressorium does not absorb any nutrients until it enters the first rice epidermal cell.Development of the appressorium and the generation of turgor pressure require storage substances,such as glycogen and liposomes,from the conidia to provide energy,which is associated with autophagy (Fosteret al.2017). Lipid and secondary metabolism is involved in the formation of theM.oryzaeappressorium (Ohet al.2008). Autophagy is a cellular process involving the degradation and recycling of cell components and it participates in conidia production,conidia germination,and appressorium formation. The mutation of genes involves in nonselective autophagy leads to a decrease of virulence inM.oryzae(Talbot and Kershaw 2009).Mutations in two autophagy genesATG1andATG8ofUstilago maydislead to morphological defects and loss of pathogenicity (Nadal and Gold2010). The growth and infection patterns ofS.turcicaandM.oryzaeare similar. Autophagy is necessary for development of the appressorium inS.turcica(Zenget al.2020).

In this study,we investigated the infection position of theS.turcicaappressorium in maize leaves to prove the importance of appressoria. Then,three inhibitors were used to investigate the effects of melanin,DNA replication,and autophagy on appressorium turgor pressure during the infection process. To analyze the sources of glycerol causing turgor pressure,the distribution and concentration of glycogen and liposomes were investigated in conidia,the germ tube,and the appressorium. This study will provide insight into the relationship between appressorium turgor and glycerol metabolism during development of theS.turcicaappressorium.

2.Materials and methods

2.1.Fungal strains,plant material,and culture conditions

TheS.turcicastrain 01-23 used in this study (deposited in the Mycotoxin and Molecular Plant Pathology Laboratory of Hebei Agricultural University,China) was grown on potato dextrose agar (PDA;2% glucose,20% potato,and 1.5% agar) at 25°C. TheS.turcicastrain was grown and stored using standard procedures,as described previously (Shenet al.2013;Liuet al.2019).

The maize inbred line B73 was used as the susceptible host forS.turcicainfection. B73 seeds were soaked in sterile water for 12 h,and then transferred to a Petri dish covered with sterile wet filter paper. The maize seeds germinated after incubating at 25°C for 36–48 h. A proper amount of fertile nutrient soil was placed in 15-cm diameter plastic flowerpots,and three maize seeds with good germination status were sown in each pot. The plants were grown in artificial climate chambers under long-day conditions (16 h light/8 h dark) at 25°C.

2.2.Enrichment of conidia and appressorium formation

To obtain the appressorium during different developmental periods,conidia were harvested from 14-day PDA cultures,and 1×104mL–1were re-suspended in sterile water. Appressorium development was inducedin vitroon slides as described previously (Maet al.2017). The conidial suspension was incubated on cellophane at 25°C in the dark before adding 5 μg mL–1TCZ,80 mmol L–1HU,or 5 mmol L–13-MA,respectively,and without any treatment as a control. The 25 μL conidial suspension droplets with/without the specified reagents were incubated on cellophane at 25°C in the dark. The formation of appressoria was observed at the indicated times using a microscope,and each treatment was repeated in three independent assays.

2.3.lnoculating the maize leaves and the infection pattern

A 20 μL aliquot of the conidia suspension (5×104mL–1) was dripped onto the surface of the maize leaf,and cultured at 25°C. Samples were taken 0,12,24,36,and 48 h after inoculation,and spores were collected for staining and morphological observations to investigate theS.turcicainfection. The invasion locations of 100 conidia were determined 36 h post-inoculation,and then the percentage that passed through epidermal cells and stomata was calculated. The experiment was repeated three times.

2.4.Trypan blue staining and microscopic observations

The inoculated leaves were soaked in Trypan blue staining solution,and boiled for 5 min at different times after inoculation. The cooked leaves were rinsed with sterile water three to five times,and transferred to saturated chloral hydrate solution for decolorization over 2 days. The decolorized leaves were observed under a microscope to investigate theS.turcicainfection process.

2.5.Turgor pressure assays

The improved incipient-cytorrhysis technology was used to determine the appressorium turgor pressure during different developmental stages (Howardet al.1991;Caoet al.2011). Conidia incubated for different times were treated with different concentrations of PEG-2000(0.230,0.225,0.220,0.215,0.210,0.200,0.195,and 0.190 mol L–1). Following a 10 min incubation in PEG solution,the proportions of collapsed and cytorrhyzed appressoria were determined in 100 cells from each PEG concentration using an optical microscope. Turgor pressure was measured at the apex of the germ tubes in samples that did not form appressoria. When 50% of the appressoria collapsed,the PEG-2000 osmolality value was used to estimate appressorium turgor pressure,i.e.,II (MPa)=α×C+β×C2,where α is ?5.2,β is 128.1,and C is molarity (Money 1989).

2.6.Glycerol concentration assays

The appressoria were collected at different developmental stages and freeze-dried. According to the kit instructions(Enzymatic determination of glycerol concentration E1012 and Applygen Technologies Inc.,Beijing,China),the lysate was added to the dried appressoria and lipase was used for inactivation at 70°C for 10 min. The solution was centrifuged at 5 000 r min–1for 5 min. The supernatant was recovered and measured at OD550nm. The glycerol concentration was calculated according to a standard curve. The curve formula wasy=0.0029x+0.0061,where they-axis is the OD value and thex-axis is glycerol concentration.

2.7.lntracellular glycogen assays

Glycogen within developing appressoria was visualized using an iodine stain consisting of 60 mg KI and 10 mg I2mL–1in distilled water (Weberet al.1998). Freshly harvested conidia at a concentration of approximately 1×104conidia mL–1were incubated on hydrophobic plastic cover slips in distilled water to investigate the different stages,including conidia,germ tube formation,appressorium development,and appressorium maturation.The dye solution was added to the incubating droplets,and brown glycogen deposits were observed under the microscope.

2.8.lntracellular liposome assays

Liposomes within developing appressoria were visualized using Oil Red-O staining (Koopmanet al.2001).AnS.turcicaconidia suspension was induced and cultured on sterile incubation membrane at 25°C. The conidia suspension induced and cultured at different times was frozen at ?80°C for 30 min,and then 20 μL of Oil Red-O staining solution was added. After standing for 24 h,the stained suspension was placed under a microscope to observe the distribution of intracellular liposomes in theS.turcicaconidia during different germination stages.

3.Results

3.1.Setosphaeria turcica relies on the appressorium to complete the infection process

The pathogen invaded the interior of the leaf by directly penetrating the maize epidermal cells (Fig.1-A–F) or the stomata (Fig.1-G and H). TheS.turcicaconidia(Fig.1-A) germinated from one end of the conidia to form a germ tube,and also germinated from both ends of the conidia and even from the intermediate cells of the conidia to form a germ tube. The conidia germinated to form germ tubes after 12 h of inoculation on isolated maize leaves (Fig.1-B). After 24 h of inoculation,the top of the germ tube gradually expanded and developed into a dome-shaped appressoria structure (Fig.1-C).After 36 h of inoculation,the hyphae penetrated the epidermal cells of the maize leaves and invaded the leaf interior (Fig.1-D). After 48 or 60 h of inoculation,the primary hyphae in the leaf interior continued to grow and produced appressorium-like structures,which invaded adjacent cells,and colonized the host (Fig.1-E and F).The pathogen invaded maize leaves from stomatain vitroafter inoculation for 24 h,and the appressorium was also observed (Fig.1-G). After inoculation for 48 h,infected hyphae grew and expanded in the stomatic chamber,and produced appressorium-like structures that invaded adjacent cells (Fig.1-H).

Fig.1 Infection process and location of the appressorium in Setosphaeria turcica. A–F,S.turcica infection of maize leaves.G and H,S.turcica infection from the maize stomata. I,differences between stomata infection and a direct infection by S.turcica. The germination and appressorium formation of 100 conidia were counted separately,as shown in the following figures. GT,germ tube;AP,appressorium;H,hyphae;S,stoma. Data represent the mean and SD of three independent experiments;asterisks indicate a significant difference (＊,P<0.05). Bars in A–E,20 μm;Bars in F,G and H,10 μm.

In this study,the percentage of epidermal cells and stomata that invaded isolated maize leaves after induction culture for 36 h was counted. A significant difference was observed between the infection rates of the pathogenic conidia from epidermal cells and stomata (Fig.1-C).The infection rate of stomata was 31.54%,while that of epidermal cells was 68.46%,which was 2.17 times that of stomata. These results show that the maize leaf blight pathogen was more likely to invade the host from the epidermal cells of maize. Therefore,S.turcicaneeds a complete appressorium structure to finish the infection process. It is unknown whether appressoria invading through the stomata also need to maintain sufficient turgor.

3.2.Effects of different inhibitors on development of the appressorium in S.turcica

To analyze the effects of melanin,DNA replication,and autophagy on development of the appressorium,three inhibitors,including a melanin inhibitor (tricyclazole,TCZ),a DNA replication inhibitor (hydroxyurea,HU),and an autophagy inhibitor (3-methyladenine,3-MA) were used to treat conidia on the incubation membrane and observe the germination process (Fig.2). HU and 3-MA completely blocked formation of the appressorium on the incubation membrane,while TCZ had no inhibitory effect. However,exposure to a genotoxic reagent and an autophagy inhibitor did not inhibit conidia germination or subsequent germ tube formation,indicating that the block was specific for initiating the appressorium.

Fig.2 Setosphaeria turcica phenotypes of the different stages in response to the inhibitors. CK,control;TCZ,tricyclazole;HU,hydroxyurea;3-MA,3-methyladenine. Bars,20 μm.

Turgor is necessary in the appressorium to allow penetration of the host epidermis and formation of infected hyphae (Kimura 2001). Thus,turgor pressure was measured during development of the appressorium under normal conditions and after the inhibitor treatments using incipient-cytorrhysis technology. HU,3-MA,and TCZ significantly reduced appressorium turgor pressure during development of the appressorium (Table 1),and HU was the most effective. TCZ inhibited the accumulation of melanin in the inner layer of the appressorium cell wall,resulting in the efflux of intracellular material and a lower turgor pressure. We speculate that autophagy and DNA replication might affect development of the appressorium by regulating the synthesis of metabolites needed for turgor,as sensitivity ofS.turcicato HU-induced genotoxic stress or 3-MA-induced inhibition of autophagy was more serious than that of melanin induced by TCZ,particularly before the appressoria developed.

Table 1 Turgor pressure of Setosphaeria turcica during different times under the different inhibitor treatments

3.3.Relationship between turgor pressure and changes in the main metabolites during S.turcica appressorium development

A high concentration of glycerol in theM.oryzaeappressorium is crucial for generating turgor to penetrate the rice epidermis (Jonget al.1997). In this study,the glycerol concentration was determined during development of theS.turcicaappressorium. The glycerol concentration in the appressorium gradually increased during development,and reached the maximum at the mature stage,which was 1.21 μg mg–1for the 01-23 strain,0.70 μg mg–1for the TCZ-treated strain,0.35 μg mg–1for the HU-treated strain,and 0.50 μg mg–1for the 3-MAtreated strain. The trend in the glycerol concentration in the appressorium was basically consistent with that of turgor pressure. When the concentration of glycerol was high,the turgor pressure of the appressorium was also high (Figs.3 and 4). This was consistent with the results of the above-mentioned tests for turgor pressure of the strains from the different treatments. Glycerol in strain 01-23 treated with TCZ tended to leak out of the appressoria because cell wall porosity changed due to the lower melanin content. However,we speculate that DNA replication and autophagy could affect glycerol concentration of theS.turcicaappressorium by regulating glycerol synthesis.

Glycogen and liposome metabolism have been proposed as major metabolic pathways to glycerol accumulation in appressoria (Thines and Talbot 2000).Therefore,the metabolism of glycogen and liposomes is closely related to the development and function of the appressorium. Glycogen and liposomes were observed and analyzed by microscopy during development of theS.turcicaappressorium.

TheS.turcicaconidia were induced and cultured on incubation membrane for different times,and then stained to observe the distribution of glycogen during germination(Fig.5). The results showed that glycogen was abundant in ungerminated conidia (Fig.5-A). Glycogen in the conidia decreased after a 6 h induced culture,when the conidia germinated and formed germ tubes in which a small amount of glycogen was detected (Fig.5-F). After 12 h of induced culture,a new appressorium formed at the apex of the germ tube,and little glycogen was observed(Fig.5-I). However,the amount of glycogen gradually increased with development of the appressorium,and a large amount of glycogen was observed in the appressorium when it matured after 24 h of induced culture (Fig.5-M). At the same time,the appressorium continued to develop and form a second appressoriumlike cell,indicating that the extra glycogen provides energy to produce the next appressorium.

Fig.3 Effects of inhibitors on glycerol concentration in Setosphaeria turcica during the same developmental period. A,conidia.B,germ tube. C,appressorium developed. D,appressorium matured. E,appressorium penetrated. The results represent three biological and experimental replicates. Bars indicate mean and SD. Means with different letters in the figure are significantly different (P<0.01). CK,control;TCZ,tricyclazole;HU,hydroxyurea;3-MA,3-methyladenine.

We germinated conidia on incubation membrane in the presence of the three inhibitors to further analyze the factors affecting glycogen distribution during development of the appressorium.

Fig.4 Effects of the same inhibitor on Setosphaeria turcica glycerol concentration at different developmental stages. A,strain 01-23. B,strain 01-23+TCZ. C,strain 01-23+HU. D,strain 01-23+3-MA. The results represent three biological and experimental replicates. Bars indicate mean and SD. Means with different letters in the figure are significantly different (P<0.01). CK,control;TCZ,tricyclazole;HU,hydroxyurea;3-MA,3-methyladenine.

The glycogen content in the germinated conidia was the same as that in untreated conidia under the TCZ treatment(Fig.5-B). A small amount of glycogen was observed in the newly formed appressorium (Fig.5-J),which gradually increased with development of the appressorium,and a large amount of glycogen was observed in mature appressoria(Fig.5-N). More glycogen accumulated in the appressorium when melanin was inhibited.

The glycogen content in the ungerminated conidia was rich under the HU treatment (Fig.5-C);after 6 h of induction culture,the conidia germinated and grew a shorter germ tube than that under the control condition(Fig.5-G). Glycogen content continuously decreased as the germ tube elongated (Fig.5-G and K). The decrease in the glycogen content indicated that inhibiting cell replication may affect glycogen synthesis to produce glycerol,and partial glycogen provided energy for elongation of the germ tubes.

When conidia germinated to the germ tube stage under the 3-MA treatment,glycogen content in the germ tube was lower than that without 3-MA,while glycogen content in the conidia was still abundant (Fig.5-E and H). After 24 h of induction culture,the germ tube continued to elongate,but no appressorium cells formed (Fig.5-P). The glycogen content in the germ tube was less than that when it was untreated,and less glycogen was in the conidia,which might be due to the failure to form an appressorium caused by blocking autophagy. As autophagy was inhibited,glycogen stored in the conidia was used to provide energy for the excess elongation of the germ tube.

Fig.5 Distribution of glycogen in Setosphaeria turcica at different developmental stages and under the inhibitor treatments. CK,control;TCZ,tricyclazole;HU,hydroxyurea;3-MA,3-methyladenine. Bar,20 μm.

Lipid droplets are a precursor of glycerol synthesis,and catabolism in the liposome is an effective means to rapidly produce glycerol (Thines and Talbot 2000).Therefore,the distribution of liposomes was observed in appressoria after different developmental stages and with and without inhibitors. The metabolism of liposomes was very active duringS.turcicaconidia germination.The distribution of intracellular liposomes during conidia germination was observed under the microscope. The results showed that ungerminated conidia contained many small lipid droplets (Fig.6-A). After 6 h of induction culture,the conidia germinated and formed germ tubes,in which many small lipid droplets were distributed(Fig.6-E). After 12 h of induction culture,the apical part of the germ tube expanded to form the primary appressorium (Fig.6-I),and the small lipid droplets in the germ tube gathered into larger lipid droplets. Many small lipid droplets were observed in the newly formed appressorium. After induction for 24 h,the appressorium continued to grow to form a second appressorium-like cell(Fig.6-M),in which small lipid droplets were observed to aggregate into a smaller number but larger volume lipid droplets. Lipid droplets in the first appressorium were still observed after the appressorium matured and even after the appressorium developed to form the second appressorium-like cell.

The turgor pressure and glycerol concentration of strain 01–23 decreased at different development stages after adding TCZ. The non-germinated conidia still contained abundant small lipid droplets (Fig.6-B),but fewer than those of the untreated conidia (Fig.6-A),and their distribution in germ tubes was also less than that of the untreated germ tubes (Fig.6-E and F). However,after 12 h of induction,liposomes in the conidia began to degrade,and fewer small lipid droplets were detected in the germ tube and appressorium than in the untreated ones (Fig.6-J and N). After 24 h of induction,the appressorium matured and large lipid droplets were observed in the germ tube (Fig.6-N). When melanin was inhibited by TCZ,the apical germ tube expanded to form the appressorium in which turgor pressure was significantly reduced,indicating that TCZ affected the accumulation of small lipid droplets.

Fig.6 Distribution of liposomes in Setosphaeria turcica at different developmental stages and under the inhibitor treatments. CK,control;TCZ,tricyclazole;HU,hydroxyurea;3-MA,3-methyladenine. Bars in A–D,10 μm;bars in E–P,20 μm.

After adding HU,the turgor pressure and glycerol concentration in conidia decreased during development,so the distribution of liposomes was observed in conidia cultured at different times. The liposome content in undifferentiated conidia was the same as normal with abundant small lipid droplets (Fig.6-C). When induced to form germ tubes,more liposomes were present in the germ tube,and lipid droplets gathered in the conidia(Fig.6-G),indicating that the lipid droplets not used to produce glycerol led to the lower turgor pressure.

After adding 3-MA,the turgor pressure and glycerol concentration of the 01-23 strain decreased during development. The distribution of lipid droplets in the strains was detected during different development stages treated with the autophagy inhibitor 3-MA. The ungerminated conidia still contained abundant lipid droplets (Fig.6-D),while the liposome content in the conidia decreased during the germ tube forming phase(Fig.6-H). After 12 h of induction,the content of lipid droplets in the conidia decreased,and small lipid droplets appeared in the germ tube (Fig.6-L). After 24 h of culture,the number of lipid droplets increased in the excessively elongated germ tube (Fig.6-P).

4.Discussion

In this study,the infection position of theS.turcicaappressorium was investigated in maize leaves. TheS.turcicainfection process of maize leaf cells depended on the complete appressorium structure,regardless of whether entry was directly through the epidermal cells or through stomata. Many fungal pathogens form specialized infection structures to penetrate the plant leaf epidermis (Dean 1997). These structures include infection cushions,appressoria,penetration pegs,and haustoria.These structures absorb nutrients and energy from the plant by adhering to the surface of the host (Tucker and Talbot 2001). Several plant pathogenic fungi develop appressoria with hard and black walls (Howard and Valent 1996),such asM.oryzaeandC.gloeosporioiles(Zhanget al.2006;Eliahuet al.2007). The appressorium tightly adheres to the plant surface and develops the infection structure that penetrates the plant cuticle (Bourett and Howard 1990;Ebbole 2007).

Glycerol produced by glycogen and liposome metabolism is the main chemical substance that maintains the high turgor pressure in the appressorium of plant pathogenic fungi (Wanget al.2012). The transfer of lipid droplets and subsequent fatty acid metabolism in the peroxisome through β-oxidation;that is,acetyl coenzyme A enters the glyoxylic acid cycle and gluconeogenesis,and the glyoxylic acid-dependent NADH reoxidizes to NADP+,which are key metabolic steps necessary for normal functioning of the appressorium (Yanget al.2012). In our study,the effects of three inhibitors (TCZ,HU,and 3-MA) on the appressorium phenotype were studied,to investigate the functions of melanin,DNA replication,and autophagy through appressorium turgor pressure,glycerol concentration,and glycogen and liposome distribution. Our findings suggested that the DNA replication and autophagy inhibitors affected the accumulation of glycogen and lipid droplets,resulting in a decrease in the glycerol concentration,and melanin prevented intracellular solutes from flowing out of the appressoria. However,inhibiting the accumulation of glycogen and lipid droplets may hinder appressorium development due to depleted energy reserves.

Cds1 regulates the S-phase during development of the appressorium inM.oryzae. ΔMocds1enters the S-phase in the presence of HU,undergoes complete mitosis,and forms the appressorium;however,after the mature ΔMocds1appressorium was treated with HU,the mutant did not enter the S-phase and produce infection nails (Osés-Ruizet al.2017). These results indicate that the S-phase is regulated by the DDR during conidia germination,while the S-phase during maturation of the appressorium infection nail is not regulated by the DDR.Further research shows that the S-phase of the second round is controlled by the magnitude of turgor pressure.The turgor pressure must reach a minimum filling threshold after the first mitosis,and the nucleus of the appressorium enters S-phase from the G1-phase. The melanin mutants ΔMorsy1and ΔMobuf1cannot develop turgor,and their cells are always in the G1-phase when they enter the second mitosis,and they cannot enter S-phase (Osés-Ruizet al.2017). After the S-phase,the G2/M-phase controls maturation of the appressorium and colonization after infection. TheS.turcicaS-phase checkpoint kinase StATR is activated to block maize infection due to genotoxic stress by inhibiting formation of the appressorium (Zenget al.2020). Our results show that the appressorium did not form inS.turcicatreated with HU to stagnate in the S-phase. At the same time,HU affected the accumulation of glycogen and lipid droplets,which resulted in low glycerol content and low turgor pressure in the germ tube.

Autophagy in theM.oryzaeconidia is necessary for the appressorium to function. Mutants lacking autophagy components cannot form functional appressoria (Claireet al.2006) and produce less turgor pressure in the appressorium (Liuet al.2007).This blocked autophagy process leads to inhibited formation of lipid droplets and the accumulation of glycogen. The autophagy process inM.oryzaecan be blocked by deleting the autophagy-related genesMgATG1(Liuet al.2007),MgATG4(Liu and Lin2008),andMgATG5(Luet al.2009),which eventually leads to the loss of pathogenicity. These results indicate that autophagy affects the turgor of the appressorium,which prevents the mutants from infecting the leaf epidermal cells effectively and they lose pathogenicity(Penget al.2011). The growth and infection pattern ofS.turcicaandM.oryzaeare similar. Autophagy is necessary for development of theM.oryzaeappressorium. Therefore,this study investigated the effect of autophagy on the development ofS.turcicaappressoria for the first time. As results,autophagy played an important role in the development of theS.turcicaappressorium,but its specific function needs to be further verified. It has been reported that glycogen is mobilized during appressorium development and that glycogen contributes to glycerol production in the appressorium (Thines and Talbot 2000). InM.oryzae,glycogen disappears from the conidia during maturation of the appressorium,but it still occurs in theS.turcicaconidia at maturation.Therefore,the metabolism of glycogen and the relationship with glycerol synthesis may be different betweenS.turcicaandM.oryzae.

5.Conclusion

In this present study,appressorium turgor pressure and glycerol concentration in the appressorium reached their highest levels at the mature stage of the appressorium under the control and after treatment with several inhibitors. The inhibitor treatments had the greatest effect on appressorium turgor pressure at this stage.The inhibitors affected the distribution and content of glycogen and liposomes in the conidia and germ tubes or between the conidia and the appressorium.Our research provides insight into the relationship between appressorium turgor pressure and glycerol content,which was affected by melanin synthesis,DNA replication,and autophagy in the developing appressorium duringS.turcicainfection.

Acknowledgements

This work was supported by the grants from the National Natural Science Foundation of China (32072370 and 31901827),the China Agriculture Research System of MOF and MARA (CARS-02-25),the Natural Science Foundation of Hebei Province,China (C2020204039 and C2018204059),and the Projects of Overseas Foundation,Hebei Province,China (C20190508).

Declaration of competing interest

The authors declare that they have no conflict of interest.