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Mol Plant Pathol
Mol Plant Pathol
10.1111/(ISSN)1364-3703
MPP
Molecular Plant Pathology
1464-6722
1364-3703
John Wiley and Sons Inc. Hoboken

10.1111/mpp.70003
MPP70003
MPP-OA-24-133.R2
Original Article
Original Article
Polyamine oxidation enzymes regulate sexual mating/filamentation and pathogenicity in Sporisorium scitamineum
Yin et al.
Yin Kai https://orcid.org/0009-0009-6998-9949
1 2
Hu Zhijian 1 2
Yuan Meiting 1 2
Chen Weidong 1 2
Bi Xinping 1 2
Cui Guobing 1 2
Liang Zhibin https://orcid.org/0000-0003-3433-0975
1 2
Deng Yi Zhen https://orcid.org/0000-0002-3572-1559
1 2 dengyz@scau.edu.cn

1 Key Laboratory for Conservation and Utilization of Subtropical Agro‐Bioresources, College of Plant Protection South China Agricultural University Guangzhou China
2 Integrative Microbiology Research Centre, Guangdong Province Key Laboratory of Microbial Signals and Disease Control South China Agricultural University Guangzhou China
* Correspondence
Yi Zhen Deng, Key Laboratory for Conservation and Utilization of Subtropical Agro‐Bioresources, College of Plant Protection, South China Agricultural University, Guangzhou 510642, China.
Email: dengyz@scau.edu.cn

05 9 2024
9 2024
25 9 10.1111/mpp.v25.9 e7000313 8 2024
24 4 2024
19 8 2024
© 2024 The Author(s). Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.
https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Abstract

Sugarcane smut fungus Sporisorium scitamineum produces polyamines putrescine (PUT), spermidine (SPD), and spermine (SPM) to regulate sexual mating/filamentous growth critical for pathogenicity. Besides de novo biosynthesis, intracellular levels of polyamines could also be modulated by oxidation. In this study, we identified two annotated polyamine oxidation enzymes (SsPAO and SsCuAO1) in S. scitamineum. Compared to the wild type (MAT‐1), the ss1paoΔ and ss1cuao1Δ mutants were defective in sporidia growth, sexual mating/filamentation, and pathogenicity. The addition of a low concentration of cAMP (0.1 mM) could partially or fully restore filamentation of ss1paoΔ × ss2paoΔ or ss1cuao1Δ × ss2cuao1Δ. cAMP biosynthesis and hydrolysis genes were differentially expressed in the ss1paoΔ × ss2paoΔ or ss1cuao1Δ × ss2cuao1Δ cultures, further supporting that SsPAO‐ or SsCuAO1‐based polyamine homeostasis regulates S. scitamineum filamentation by affecting the cAMP/PKA signalling pathway. During early infection, PUT promotes, while SPD inhibits, the accumulation of reactive oxygen species (ROS) in sugarcane, therefore modulating redox homeostasis at the smut fungus–sugarcane interface. Autophagy induction was found to be enhanced in the ss1paoΔ mutant and reduced in the ss1cuao1Δ mutant. Exogenous addition of cAMP, PUT, SPD, or SPM at low concentration promoted autophagy activity under a non‐inductive condition (rich medium), suggesting a cross‐talk between polyamines and cAMP signalling in regulating autophagy in S. scitamineum. Overall, our work proves that SsPAO‐ and SsCuAO1‐mediated intracellular polyamines affect intracellular redox balance and thus play a role in growth, sexual mating/filamentation, and pathogenicity of S. scitamineum.

Polyamines homeostasis control dimorphic switch of sugarcane smut fungus via modulation of autophagy and intracellular reactive oxygen species levels.

copper amine oxidase (CuAO)
pathogenesis
polyamine oxidase (PAO)
redox
sexual mating/filamentation
Sporisorium scitamineum
National Natural Science Foundation of China 10.13039/501100001809 32102156 U23A20148 source-schema-version-number2.0
cover-dateSeptember 2024
details-of-publishers-convertorConverter:WILEY_ML3GV2_TO_JATSPMC version:6.4.8 mode:remove_FC converted:05.09.2024
Yin, K. , Hu, Z. , Yuan, M. , Chen, W. , Bi, X. , Cui, G. et al. (2024) Polyamine oxidation enzymes regulate sexual mating/filamentation and pathogenicity in Sporisorium scitamineum . Molecular Plant Pathology, 25 , e70003. Available from: 10.1111/mpp.70003
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pmc1 INTRODUCTION

The sugarcane smut disease that seriously affects sugarcane yield and results in economic losses is caused by the dimorphic fungus Sporisorium scitamineum, which has three lifestyles: haploid yeast‐type sporidia (unable to infect the host) of two opposite mating types (MAT‐1 and MAT‐2), dikaryotic hyphae, and diploid teliospores (Piepenbring et al., 2002; Yan et al., 2016). Sexual mating between haploid sporidia of different mating types gives rise to dikaryotic hyphae capable of infecting host canes (Yan et al., 2016). The dikaryotic hyphae grow in the apoplastic space between the plant cells of young apical tissues for weeks to months, before formation of teliospores wrapped by plant tissues in a ‘whip’ shape, the typical symptom of sugarcane smut disease (Lu et al., 2021). Therefore, sexual mating/filamentation and teliospore formation play a key role in S. scitamineum pathogenicity.

Polyamines are small organic polycations with primary and secondary amino groups. They are widely found in all living organisms and participate in a variety of biological processes (Rocha & Wilson, 2019). The most common polyamines are diamine putrescine (PUT), triamine spermidine (SPD), and tetramine spermine (SPM), although a large number of algae, fungi, and bacterial species do not contain SPM (Hamana & Matsuzaki, 1982; Tavladoraki et al., 2012; Valdes‐Santiago et al., 2010). Polyamine biosynthesis, conjugation, catabolism, acetylation, and transport are strictly regulated to maintain the homeostasis of the polyamine pool and intracellular environment (Tavladoraki et al., 2012). Insufficient or excessive levels of polyamines are detrimental to cell growth and development and may trigger a series of reactions leading to cell death (Gerlin et al., 2021; Kusano et al., 2008). Polyamine metabolism pathways in plants and animals have been extensively studied (Casero & Marton, 2007; Efrose et al., 2008; Kusano et al., 2008). Our previous work elucidated the role of the polyamine biosynthesis pathway in S. scitamineum (Yin et al., 2024). Besides de novo biosynthesis of polyamines, intracellular polyamine homeostasis can be maintained by oxidization and/or acetylation. Amine oxidases catalyse the oxidative de‐amination of polyamines at their primary or secondary amino‐groups, which can be classified according to their cofactors as flavine‐containing polyamine oxidases or copper‐containing amine oxidases (CuAOs) (Cona et al., 2006; Planas‐Portell et al., 2013). Flavine‐containing amine oxidases include acetylated polyamine oxidase (APAO), polyamine oxidase (PAO), monoamine oxidase (MAO), and SPM oxidase (SMO) (McGrath et al., 2011; Miller‐Fleming et al., 2015). Two types of quinone cofactors exist in CuAOs, namely 2,4,5‐trihydroxyphenylalanine quinone (TPQ) and lysine tyrosyl quinone (LTQ) (McGrath et al., 2011).

Polyamine oxidases from plants and bacteria can oxidize both SPM and SPD, resulting in products including 4‐aminobutyraldehyde, 1,3‐diaminopropane, and hydrogen peroxide (H2O2), a type of reactive oxygen species (ROS) causing oxidative stress and damage to cellular components. However, the production of ROS during polyamine oxidation can also activate the antioxidative response in cells (Murray Stewart et al., 2018). The first characterized PAO, maize PAO (ZmPAO), is an apoplastic enzyme (Tavladoraki et al., 1998). AtPAO1, AtPAO2, AtPAO3, and AtPAO4 of Arabidopsis thaliana preferentially oxidize the free form of SPD, SPM, T‐SPM (thermospermine), or Nor‐SPM (norspermine), to produce 1,3‐aminopropanal, H2O2, PUT or SPD (Fincato et al., 2011; Kamada‐Nobusada et al., 2008; Moschou et al., 2008; Ono et al., 2012; Tavladoraki et al., 2006, 2016). Besides the aforementioned substrates, AtPAO5 oxidizes also N 1‐acetyl‐SPM (Ahou et al., 2014; Liu et al., 2014), and regulates A. thaliana growth (Ahou et al., 2014; Kim et al., 2014; Tavladoraki et al., 2016). Catabolism of polyamines in mammals is sequentially catalysed by SMO, which specifically oxidizes SPM to produce SPD, H2O2, and 1,3‐aminopropanal, and then by inducible SPM/SPD N1‐acetyltransferase (SSAT, which transfers an acetyl group to SPM or SPD) or the constitutively expressed N1‐acetylpolyamine oxidase (APAO) (oxidase acetyl‐SPM and acetyl‐SPD) (Cervelli et al., 2013, 2016; Polticelli et al., 2012). SSAT‐mediated acetylation can reduce the positive charge of polyamines, thereby preventing their interaction with other molecules (Seiler, 1987). The acetylated SPD or SPM can be excreted or oxidized by APAO, to become SPD or SPM again (Wallace et al., 2003). These cyclic reactions allow the cells to rapidly regulate the intracellular concentration of SPD or SPM (Seiler, 1987). Saccharomyces cerevisiae PAO (named Fms1) is able to oxidize SPM and acetyl‐SPM, producing SPD and 1,3‐aminopropanal (Huang et al., 2005; Landry & Sternglanz, 2003; Tavladoraki et al., 1998). CuAOs from microbes, animals, and plants catalyse the oxidative deamination of primary amines in polyamines, but no activity has been found against secondary or tertiary amines of PUT and SPD, as opposed to PAOs (Largeron et al., 2010; Shepard & Dooley, 2015). CuAO‐catalysed oxidation forms the corresponding aldehydes, with concomitant reduction of molecular oxygen to H2O2 (Parsons et al., 1995). Amino oxidases including PAOs and CuAOs contribute to polyamines and cellular redox homeostasis in plant cells during growth, development, and response to abiotic or biotic stresses (Angelini et al., 2010; Cona et al., 2006; Rea et al., 2004; Walters, 2003; Wu et al., 2003). However, biological function and regulatory mechanism of oxidation of polyamines has not been reported in S. scitamineum.

In this study, we identified SsPAO and SsCuAO1 in S. scitamineum and characterized their biological functions by reverse genetics. Deletion of SsPAO or SsCuAO1 led to reduced sporidia growth, reduced sexual mating/filamentation, and weak or loss of pathogenicity in S. scitamineum. Our results further demonstrate that modulation of intracellular polyamines by SsPAO or SsCuAO1 could regulate the cAMP‐PKA pathway, which may affect autophagy in S. scitamineum. Overall, our work reveals that SsPAO and SsCuAO1 regulate sexual mating/filamentation and pathogenicity in S. scitamineum, probably by affecting turnover of intracellular polyamines and the consequent redox homeostasis.

2 RESULTS

2.1 SsPAO and SsCuAO1 modulate polyamine levels

To identify enzymes catalysing oxidation of polyamines in S. scitamineum, we used the FMS1‐polyamine oxidase PAO (XP_011392213.1) (Valdes‐Santiago et al., 2010) and the annotated copper amine oxidase 1 CuAO1 (XP_011390076.1) in Ustilago maydis, as the queries to perform a BLASTP search on the NCBI website (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome). CDU22952.1 (encoded by SPSC_01582) was identified as an orthologue of U. maydis PAO, and CDR87209.1 (encoded by SPSC_00335) was orthologous to U. maydis CuAO1. We named these two proteins as SsPAO and SsCuAO1, respectively. Phylogenetic analysis using SsPAO and SsCuAO1 and their orthologues from smut fungi or ascomycetes (Table S1) showed that these two proteins were closest to the orthologues in Sporisorium reilianum, forming a clade within smut fungi and separating from ascomycetes (Figure S1a,b). Based on prediction by the SmartBLAST tool (https://blast.ncbi.nlm.nih.gov/smartblast/), SsPAO contains a conserved amino_oxidase domain (PF01593) (Figure S1a), and SsCuAO1 contains a copper amine oxidase N2‐terminal domain (PF02727) and a copper amine oxidase catalytic domain (PF01179) (Figure S1b).

By homologous recombination, we generated deletion mutants of the SsPAO or SsCuAO1 genes in both MAT‐1 and MAT‐2 backgrounds, as verified by Southern blotting (Figure S2a,b). We named the single‐deletion mutants as ss1paoΔ (in MAT‐1 background) and ss2paoΔ (in MAT‐2 background), ss1cuao1Δ (in MAT‐1 background), and ss2cuao1Δ (in MAT‐2 background). Meanwhile, the genetic complementation strains were generated, namely Ss1PAO‐COM, Ss2PAO‐COM, Ss1CuAO1‐COM, and Ss2CuAO1‐COM, and were verified by PCR (Figure S2c,d). Fluorescence microscopy analysis revealed that the fusion proteins GFP‐SsPAO and SsCuAO1‐RFP used for genetic complementation were localized in the cytoplasm (Figure S2e,f). For all the deletion mutants and complementation strains, at least two independent strains were obtained and used for the following assessments.

To assess the possible function of SsPAO and SsCuAO1 in polyamine metabolism, we measured the intracellular and extracellular levels of PUT, SPD, and SPM in the wild‐type (WT) (MAT‐1), ss1paoΔ, and ss1cuao1Δ mutant by liquid chromatography‐mass spectrometry (LC–MS) analysis, following an established protocol (Yin et al., 2024). The intracellular level of PUT was significantly higher in ss1paoΔ (p < 0.05) and ss1cuao1Δ (p < 0.001) compared to MAT‐1, but there was no significant difference in intracellular SPD or SPM level (Figure 1a). The extracellular level of SPM was significantly higher in ss1cuao1Δ (p < 0.01) compared to MAT‐1 (Figure 1a). We further evaluated the effect of polyamines on transcription of SsPAO and SsCuAO1 genes. Liquid minimal medium (MM)‐cultured MAT‐1 sporidia were treated with PUT (1 mM), SPD (1 mM), or SPM (1 mM), for 90 min, before total RNA extraction for reverse transcription‐quantitative PCR (RT‐qPCR) analysis. The result showed that PUT treatment caused down‐regulation of SsCuAO1 and polyamine biosynthetic genes (SsAGA, SsAGT, SsODC, SsSAMDC, and SsSPDS/SPMS) (Figure 1b). In contrast, SPD treatment up‐regulated transcription of SsPAO, SsCuAO1, and SsAGA, but down‐regulated SsSAMDC and SsSPDS/SPMS transcription (Figure 1b). SPM treatment up‐regulated SsCuAO1 but down‐regulated SsPAO and the biosynthetic genes (Figure 1b). Our previous study showed that SsCuAO1 was up‐regulated in the ss1agaΔ mutant (Yin et al., 2024), probably as a response to elevated PUT or SPM levels. Consistent with the results obtained here, this suggests that SsPAO may be involved in SPD oxidation to produce PUT, and SsCuAO1 may oxidize SPM and/or SPD to produce SPD and PUT, respectively.

FIGURE 1 SsPAO and SsCuAO1 contribute to maintaining polyamine levels in Sporisorium scitamineum. (a) Measurement of intracellular and extracellular polyamines of MAT‐1, ss1paoΔ or ss1cuao1Δ. Fresh haploid sporidia were cultured in liquid YePS medium for 12 h, washed twice with double‐distilled water, and adjusted to OD600 = 1.0. Polyamine contents were measured from crude extracts of fungal cells or from the supernatant of culture medium, by LC–MS. Bar chart depicts the mean ± SE derived from three independent biological repeats, each containing three replications (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, versus MAT‐1. (b) Transcriptional profiling of SsPAO, SsCuAO1 and polyamine biosynthesis genes in MAT‐1 after exogenous supply of 1 mM putrescine (PUT), spermidine (SPD) or spermine (SPM). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, versus control (untreated cell). (c) Transcriptional profiling of polyamine biosynthesis genes and ‘a locus’ genes (SsMFA1 and SsPRA1) in ss1paoΔ and ss1cuao1Δ, compared to MAT‐1. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, versus MAT‐1. (d) Transcriptional profiling of SsPAO or SsCuAO1 genes during early stage of sugarcane infection. At 0, 3 or 7 days post‐inoculation (dpi), approximately 3 cm stems of the inoculated site were sampled for RNA extraction. ***p < 0.001, ****p < 0.0001, versus SsPAO (0 dpi). For (b–d), the relative gene expression levels were calculated according to the −ΔΔC t method (Livak & Schmittgen, 2001), with Actin as an internal control. Bar chart depicts the mean ± SE derived from three independent biological repeats, each containing three replications (n = 3).

During sexual mating/filamentation, relative expression levels of polyamine biosynthesis genes and the ‘a locus’ genes (pheromone MFA1 and pheromone receptor PRA1) were examined in these two mutants compared to WT MAT‐1 × MAT‐2. Results showed that SsODC, SsSAMDC, SsSPDS/SPMS, SsMFA1, and SsPRA1 were down‐regulated in the ss1paoΔ mutant. On the other hand, SsSAMDC and SsSPDS/SPMS were down‐regulated while SsMFA1 and SsPRA1 were up‐regulated in the ss1cuao1Δ mutant (Figure 1c). Subsequently, we measured the relative transcription levels of SsPAO and SsCuAO1 during the early stage of host infection, when changes in intracellular and/or extracellular levels of polyamines in sugarcane–S. scitamineum interface were expected. The results showed that SsPAO and SsCuAO1 were down‐regulated at 3 and 7 days post‐inoculation (dpi) (Figure 1d). We infer that the pathogen needs to maintain a high level of polyamines, especially for SPD as reported (Yin et al., 2024), during host infection, thus down‐regulating SsPAO and SsCuAO1 at this stage.

Overall, our results showed that SsPAO and SsCuAO1 modulate polyamine levels in S. scitamineum during sexual mating/filamentation and host infection stages.

2.2 SsPAO and SsCuAO1 are required for sporidia growth, sexual mating, and filamentation

Next, we assessed the yeast‐like growth and development of sporidia of ss1paoΔ and ss1cuao1Δ mutants in comparison to MAT‐1. Under the microscope, we noticed that ss1paoΔ sporidia were significantly (p < 0.0001) longer than MAT‐1, but there was no significant difference in ss1cuao1Δ compared with MAT‐1 (Figure 2a,b). Growth curve analysis showed that the logarithmic growth phase of ss1paoΔ and ss1cuao1Δ significantly lagged behind that of MAT‐1 (Figure S3a,b). Addition of PUT (1 mM), SPD (1 mM) or SPM (0.1 mM) did not affect the growth of MAT‐1 or ss1paoΔ (Figure S3a,b), whereas SPM significantly suppressed the growth of ss1cuao1Δ sporidia (Figure S3b). There was no significant difference in colony morphology of ss1paoΔ and MAT‐1 when spotted on potato dextrose agar (PDA) or minimal medium (MM) in the presence of PUT (1 mM), SPD (1 mM), or SPM (0.1 mM), whereas SPM significantly suppressed growth of ss1cuao1Δ sporidia on solid MM (Figure S3c). Overall, loss of SsPAO or SsCuAO1 led to a lag in sporidia growth of S. scitamineum, and ss1cuao1Δ seemed more sensitive to exogenous SPM treatment, further supporting that SsCuAO1, but not SsPAO, may be involved in SPM oxidation.

FIGURE 2 SsPAO and SsCuAO1 are required for sporidia growth and sexual mating/filamentation. (a) Microscopic observation of sporidia morphology of MAT‐1, ss1paoΔ and ss1cuao1Δ. The fresh haploid sporidia were cultured in liquid YePS medium for 16 h and subject to microscopic observation and imaging. Scale bar = 10 μm. (b) Quantitative analysis of the length of MAT‐1, ss1paoΔ and ss1cuao1Δ. n = 20. ****p < 0.0001, versus MAT‐1. (c) Mating assay of wild‐type (WT) (MAT‐1 × MAT‐2), sspaoΔ (ss1paoΔ × ss2paoΔ) and sscuao1Δ (ss1cuao1Δ × ss2cuao1Δ). Fresh haploid sporidia were cultured in liquid YePS medium for 12 h, washed twice with double‐distilled water, and adjusted to OD600 = 1.0. Equal volume of the compatible WT strain or mutants of opposite mating types were mixed and spotted onto potato dextrose agar (PDA), containing or without (control) putrescine (PUT, 0.1 mM), spermidine (SPD, 1 mM) or spermine (SPM, 0.1 mM). The mating cultures were incubated at 28°C for 2–3 days, before observation and imaging. Scale bar = 1 mm. (d) Mating assay of WT (MAT‐1 × MAT‐2), sspaoΔ (ss1paoΔ × ss2paoΔ), SsPAO‐COM (Ss1PAO‐COM × Ss2PAO‐COM), sscuao1Δ (ss1cuao1Δ × ss2cuao1Δ), and SsCuAO1‐COM (Ss1CuAO1‐COM × Ss2CuAO1‐COM). Fresh haploid sporidia were cultured in liquid YePS medium for 12 h, washed twice with double‐distilled water, and adjusted to OD600 = 1.0. Equal volume of the compatible WT strain or mutants of opposite mating types were mixed and spotted onto potato dextrose agar. The mating cultures were incubated at 28°C for 2–3 days, before observation and imaging. Scale bar = 1 mm. (e) Transcriptional profiling of sexual mating genes in ss1paoΔ × ss2paoΔ and ss1cuao1Δ × ss2cuao1Δ mutants, in comparison to MAT‐1 × MAT‐2. Bar chart depicts the mean ± SE derived from three independent biological repeats, each containing three replications (n = 3). *p < 0.05, **p < 0.01, ****p < 0.0001, versus MAT‐1 × MAT‐2.

Dikaryotic hyphal growth after sexual mating was significantly reduced in ss1paoΔ × ss2paoΔ and ss1cuao1Δ × ss2cuao1Δ compared to MAT‐1 × MAT‐2 (Figure 2c). Genetic complementation of SsPAO or SsCuAO1 fully restored dikaryotic hyphae in the respective mutants (Figure 2d), confirming that these two genes were indeed required for S. scitamineum sexual mating/filamentation. Exogenous addition of a low concentration of PUT (0.1 mM) sightly promoted filamentation of MAT‐1 × MAT‐2 and ss1cuao1Δ × ss2cuao1Δ, whereas SPD (1 mM) or SPM (0.1 mM) markedly restored sexual mating/filamentation of ss1paoΔ × ss2paoΔ and ss1cuao1Δ × ss2cuao1Δ (Figure 2c). RT‐qPCR analysis showed that the relative expression levels of the genes involved in sexual mating/filamentation, including SsKPP2, SsbE, SsbW, SsPRF1, and SsROP1, were all significantly down‐regulated in ss1paoΔ × ss2paoΔ, while SsGPA3, SsUAC1, SsADR1, SsFUZ7, SsKPP4, SsKPP2, SsbE, SsbW, SsPRF1, SsROP1, and SsCRK1 were all down‐regulated in ss1cuao1Δ × ss2cuao1Δ (Figure 2e). We infer that increased intracellular PUT content in ss1paoΔ × ss2paoΔ and ss1cuao1Δ × ss2cuao1Δ could be responsible for the defective sexual mating/filamentation.

Overall, we conclude that SsPAO and SsCuAO1 are required for the sporidia growth, sexual mating and filamentation of S. scitamineum.

2.3 SsPAO and SsCuAO1 are required for S. scitamineum pathogenicity

We further assessed the role of SsPAO and SsCuAO1 in S. scitamineum pathogenicity. Sugarcane seedlings were inoculated with MAT‐1 × MAT‐2, ss1paoΔ × ss2paoΔ, or ss1cuao1Δ × ss2cuao1Δ sporidia and disease symptoms were documented at 3–6 months post‐inoculation. The disease incidence (appearance of whip structures) of wild‐type infected plants was 40.8 ± 8.2% (n = 20), and 9.2 ± 5.9% (n = 20) for those infected by ss1paoΔ × ss2paoΔ. No black whip symptom was found in the seedlings infected by ss1cuao1Δ × ss2cuao1Δ (n = 20) (Figure 3a,b). We measured the average plant height as an indicator of smut fungus infection. The mutant‐infected plants or noninfected (inoculated with double‐distilled water as a blank control) plants were significantly taller than those infected by MAT‐1 × MAT‐2 (Figure 3c). Teliospores of MAT‐1 × MAT‐2 or ss1paoΔ × ss2paoΔ collected from the diseased plants, as verified by PCR amplification (Figure S4a), were assessed for germination. After 30 days of incubation under a cyclic light/dark condition, the promycelia formed from wild‐type teliospores displayed obvious and dense light and dark bands (width of 2.44 ± 0.45 mm), while the sspaoΔ promycelial colony appeared looser (width of 3.90 ± 0.29 mm; Figure 3d,e).

FIGURE 3 SsPAO and SsCuAO1 are required for Sporisorium scitamineum pathogenicity. (a) Pathogenicity assay. One millilitre of mixed sporidia of MAT‐1 × MAT‐2, ss1paoΔ × ss2paoΔ or ss1cuao1Δ × ss2cuao1Δ was injected into the seedlings of sugarcane cultivar ROC22, with double‐distilled water serving as a blank control. Each treatment contained at least 20 seedlings (n = 20). Red boxed region is shown enlarged, showing the ‘black whip’ symptoms appearing on top of seedlings infected by the wild type (WT) (MAT‐1 × MAT‐2). (b) Incidence rate of disease symptom in the seedlings infected by WT (MAT‐1 × MAT‐2), sspaoΔ (ss1paoΔ × ss2paoΔ) or sscuao1Δ (ss1cuao1Δ × ss2cuao1Δ). *p < 0.05, **p < 0.01, versus WT (MAT‐1 × MAT‐2). (c) Quantitative analysis of the heights of infected seedlings. n = 34, 21, 20, 23, respectively. ***p < 0.001, ****p < 0.0001, versus WT (MAT‐1 × MAT‐2). (d) WT or sspaoΔ teliospores were allowed to germinate under a cyclic light/dark (24 h/24 h) condition for 30 days. The resultant promycelial colonies were photographed. Scale bar = 9 mm. (e) Quantitative analysis of the band width of promycelial colonies from WT or sspaoΔ teliospores. Bar chart depicts the mean ± SD derived from three independent biological repeats, each containing five replications (n = 5). ***p < 0.001, versus WT. (f) Analysis of fungal biomass by quantitative PCR. Mixed sporidia of MAT‐1 × MAT‐2, ss1paoΔ × ss2paoΔ or ss1cuao1Δ × ss2cuao1Δ were injected into the sugarcane seedlings, and the infected stems (about 3 cm) were sampled for DNA extraction at 3 and 7 days post‐inoculation (dpi). Fungal Actin gene and sugarcane GAPDH gene served as internal controls. Bar chart depicts the mean ± SE derived from three independent biological repeats, each containing three replications (n = 3). ***p < 0.001, versus WT (MAT‐1 × MAT‐2).

Next, we measured the fungal biomass in the infected seedlings at 3 and 7 dpi. The biomass of ss1paoΔ × ss2paoΔ or ss1cuao1Δ × ss2cuao1Δ mutants was comparable to MAT‐1 × MAT‐2 at 3 dpi (Figure 3f), further supporting that the oxidation of polyamines may not be needed at the early stage of host infection. At 7 dpi, biomass of the WT strain became almost 2‐fold of that at 3 dpi, while ss1paoΔ × ss2paoΔ or ss1cuao1Δ × ss2cuao1Δ did not seem obviously increased in the infected plants (Figure 3f). This indicates that the mutants were less capable of adapting to the host environment and/or quickly proliferating in planta.

Overall, we conclude that SsPAO‐ and SsCuAO1‐mediated polyamine homeostasis plays an important role in host colonization and pathogenicity of S. scitamineum.

2.4 Polyamine oxidation affects cAMP‐PKA pathway

It has been established that cAMP signalling pathway regulates sexual mating and filamentation in smut fungi including S. scitamineum (Chang et al., 2019; Kaffarnik et al., 2003). We therefore assessed the effect of cAMP on sexual mating/filamentation of WT, sspaoΔ or sscuao1Δ. A low concentration cAMP (0.1 mM) fully restored filamentation of sspaoΔ or sscuao1Δ (Figure 4a). In contrast, a high concentration cAMP (1 mM) inhibited filamentation of WT but was less inhibitory to filamentation of sscuao1Δ (Figure 4a). cAMP, of either low or high concentration, did not affect sporidia growth of MAT‐1, ss1paoΔ or ss1cuao1Δ (Figure S4b). However, the intracellular contents of cAMP were comparable in ss1paoΔ or ss1cuao1Δ to MAT‐1 sporidia, or in ss1paoΔ × ss2paoΔ or ss1cuao1Δ × ss2cuao1Δ to MAT‐1 × MAT‐2 (Figure 4b). By RT‐qPCR, we assessed transcriptional levels of genes encoding enzymes for cAMP synthesis (adenylate cyclase, UAC1) or hydrolysis (3′,5′‐cyclic‐AMP phosphodiesterase, PDE1) in WT (MAT‐1 × MAT‐2), sspaoΔ (ss1paoΔ × ss2paoΔ) or sscuao1Δ (ss1cuao1Δ × ss2cuao1Δ). The results showed that SsUAC1 was significantly down‐regulated in ss1cuao1Δ × ss2cuao1Δ and up‐regulated in ss1paoΔ × ss2paoΔ compared to MAT‐1 × MAT‐2, whereas SsPDE1 was significantly down‐regulated in ss1cuao1Δ × ss2cuao1Δ (Figure 4c).

FIGURE 4 Assessment of cAMP‐PKA pathway in sspaoΔ and sscuao1Δ mutants. (a) cAMP differentially regulates sexual mating/filamentation in sspaoΔ and sscuao1Δ mutants. The mating cultures were allowed to grow on potato dextrose agar (PDA) containing (0.1 or 1 mM) or without (control) cAMP, at 28°C for 2–3 days, before observation and imaging. Scale bar = 1 mm. (b) Measurement of intracellular cAMP level of MAT‐1, ss1paoΔ, ss1cuao1Δ, wild‐type (WT) (MAT‐1 × MAT‐2), sspaoΔ (ss1paoΔ × ss2paoΔ) and sscuao1Δ (ss1cuao1Δ × ss2cuao1Δ). Sporidia cultured in liquid YePS medium for 24 h before measurement of the intracellular cAMP level. For mating cultures, sporidia were cultured in liquid YePS medium for 12 h, washed twice with double‐distilled water, and adjusted to OD600 = 1.0. Equal volumes of the compatible WT strain or mutants of opposite mating types were mixed and spotted onto solid YePS medium. The mating cultures were incubated at 28°C for 2–3 days, before measurement of the intracellular cAMP level. The cAMP level was measured from crude extracts, by a Complete cAMP ELISA kit. Bar chart depicts the mean ± SE derived from three independent biological repeats, each containing three replications (n = 3). (c) Transcriptional profile of SsUAC1 and SsPDE1 in WT (MAT‐1 × MAT‐2), sspaoΔ (ss1paoΔ × ss2paoΔ) and sscuao1Δ (ss1cuao1Δ × ss2cuao1Δ). Bar chart depicts the mean ± SE derived from three independent biological repeats, each containing three replications (n = 3). *p < 0.05, ****p < 0.0001, versus WT (MAT‐1 × MAT‐2). (d) Mating assay of WT (MAT‐1 × MAT‐2), ss1paoΔ × MAT‐2, GAPD::SsPRF1 (ss1paoΔ) × MAT‐2, ss1cuao1Δ × MAT‐2 and GAPD::SsPRF1 (ss1cuao1Δ) × MAT‐2. Scale bar = 1 mm.

We further constructed a reintegrated homologous fragment for overexpression of PRF1 (pheromone response factor 1) gene, which is known as a central transcriptional regulator in sexual mating/filamentation in S. scitamineum (Zhu et al., 2019). By homologous recombination we inserted the SsPRF1 overexpression fragments in the genome of the ss1paoΔ and ss1cuao1Δ mutants, resulting in GAPD::SsPRF1 (ss1paoΔ) and GAPD::SsPRF1 (ss1cuao1Δ) strains as verified by RT‐qPCR (Figure S4c). Filamentation was slightly promoted in GAPD::SsPRF1 (ss1cuao1Δ) × MAT‐2, but not in GAPD::SsPRF1 (ss1paoΔ) × MAT‐2 (Figure 4d).

Taken together, we conclude that deletion of SsCuAO1 leads to the imbalance of intracellular polyamines, which affects cAMP‐PKA signalling, but not cAMP level that caused defective sexual mating/filamentation.

2.5 SsPAO and SsCuAO1 regulate autophagy

It has been reported that SPD induces autophagy in yeast, and PUT stimulates autophagy in the phytopathogenic fungus Colletotrichum higginsianum (Madeo et al., 2010; Yan et al., 2022). We wondered whether cAMP and/or polyamines regulate autophagy in S. scitamineum. Therefore, we used GFP‐SsAtg8 as a marker to monitor non‐selective autophagy in MAT‐1. Both the full‐length GFP‐SsAtg8 fusion protein (42 kDa) and the band corresponding to free GFP peptide (27 kDa) could be detected by anti‐GFP antibody. When the fungal cells were cultured in rich medium (YePS), usually only the full‐length GFP‐SsAtg8 band was detected, whereas the free GFP peptide was detected and accumulated in the fungal cells cultured under nitrogen starvation (on MM−N) conditions (Figure 5a,b). This verifies that the strain GFP‐SsATG8:MAT‐1 could be used for monitoring autophagy activity in S. scitamineum. The ratio of GFP:(GFP + GFP‐ATG8) was used as an indicator of autophagy level. When treated with PUT, SPD, SPM, or cAMP, the GFP:(GFP + GFP‐ATG8) ratio was increased under both YePS and MM−N culture conditions (Figure 5a,b), indicating that polyamines and cAMP were capable of enhancing autophagy activity, especially under rich medium conditions.

FIGURE 5 SsPAO and SsCuAO1 regulate autophagy. (a) Fungal cells were cultured in liquid YePS medium (rich medium) for 16 h, then transferred to fresh liquid YePS or MM−N (nitrogen starvation), with or without supplement of putrescine (PUT, 0.1 mM or 1 mM), spermidine (SPD, 0.1 mM or 1 mM), spermine (SPM, 0.1 mM or 1 mM), or cAMP (0.1 mM or 1 mM), for further incubation for 4 h. Immunoblotting analysis with anti‐GFP antibody was performed to evaluate autophagy activity. Coomassie brilliant blue (CBB) staining served as loading control. The GFP:(GFP + GFP‐ATG8) ratio was calculated based on pixel intensity of the detected bands. Three biological replicates were performed with similar results. (b) Quantification of the autophagy flux based on area and pixel intensity of the detected protein bands, measured by using ImageJ software (v. 1.53 k). Bar chart depicts the mean ± SE derived from three independent biological repeats, each containing three replications (n = 3). **p < 0.01, ***p < 0.001, ****p < 0.0001, versus control condition (YePS). (c) MAT‐1, ss1paoΔ or ss1cuao1Δ sporidia were grown in YePS or MM−N, with or without exogenous addition of PUT or SPD, before total protein extraction. Full‐length or PE‐conjugated form of SsAtg8 were detected using anti‐ATG8 antibody. The ratio of ATG8‐PE:(ATG8 + ATG8‐PE) was calculated based on pixel intensity of the detected bands. Three biological replicates were performed with similar results. (d) Quantification of the autophagy flux based on pixel intensity. Bar chart depicts the mean ± SE derived from three independent biological repeats, each containing three replications (n = 3). ***p < 0.001, versus control condition (YePS).

We further assessed autophagy activity in MAT‐1, ss1paoΔ and ss1cuao1Δ, using an anti‐Atg8 antibody. The full‐length SsAtg8 peptide could be detected as a band of approximately 15 kDa, and the band slightly smaller than full‐length SsAtg8 corresponds to the PE‐conjugated variant, SsAtg8‐PE (Figure 5c), reflecting autophagy induction (Kabeya et al., 2000). When grown in YePS medium, the MAT‐1 strain contained lower amounts of SsAtg8‐PE (Figure 5c,d), suggesting that a rich nutrient environment repressed autophagy induction. When grown in MM−N medium, the MAT‐1 strain exhibited increased amounts of SsAtg8‐PE (Figure 5c,d). However, both YePS‐ or MM‐N‐cultured ss1paoΔ mutant displayed a higher level of SsAtg8‐PE (Figure 5c,d), indicating an enhanced level of autophagy induction. In contrast, lower amounts of SsAtg8‐PE were detected in the ss1cuao1Δ mutant under autophagy‐induction conditions (MM−N; Figure 5c,d). Exogenous addition of PUT or SPD caused a decrease in the ATG8‐PE:(ATG8 + ATG8‐PE) ratio in the ss1paoΔ mutant compared to the untreated condition (Figure 5c,d). In contrast, exogenous PUT or SPD led to an enhanced ATG8‐PE:(ATG8 + ATG8‐PE) ratio in ss1cuao1Δ compared to untreated condition (Figure 5c,d). These results indicate that autophagy activity is regulated by SsPAO and SsCuAO1, not just by their function on polyamine homeostasis but may involve other mechanisms.

We also examined expression levels of autophagy genes SsATG1, SsATG13, SsATG17, and SsATG8, in MAT‐1, ss1paoΔ and ss1cuao1Δ, cultured in YePS or MM−N. All the tested genes were up‐regulated in MAT‐1 in response to nitrogen starvation (MM−N) (Figure S4d). Transcriptional levels of SsATG8 were higher in ss1paoΔ under MM−N compared to YePS conditions. However, transcriptional levels of SsATG8 were not significantly different in ss1cuao1Δ under MM−N compared to YePS conditions (Figure S4d). This result suggests that SsPAO may negatively, and SsCuAO1 positively, regulate autophagy in S. scitamineum. Furthermore, treatment with rapamycin (55 nM), an established autophagy inducer (Yoo et al., 2019), slightly suppressed ss1paoΔ filamentation but slightly promoted ss1cuao1Δ filamentation (Figure S4e). This also indicates that the basal level of autophagy may be higher in the ss1paoΔ mutant and lower in the ss1cuao1Δ mutant.

Overall, our results showed that SsPAO‐ and SsCuAO1‐mediated polyamine metabolism differentially regulates autophagy activity in S. scitamineum.

2.6 SsPAO and SsCuAO1 are involved in stress tolerance

Stress tolerance analysis showed that ss1paoΔ and ss1cuao1Δ mutants were more sensitive to Congo Red (CR, 1 mM, cell wall stress), but showed no difference compared with MAT‐1 when grown on media containing NaCl (500 mM, osmotic stress), sorbitol (1 M, osmotic stress), or SDS (0.1 mM, cell wall stress) (Figure S5a). The mating ss1paoΔ × ss2paoΔ or ss1cuao1Δ × ss2cuao1Δ cultures hardly produce dimorphic hyphae under stressful conditions, whereas MAT‐1 × MAT‐2 could also produce short dikaryotic hyphae (Figure 6a).

FIGURE 6 SsPAO and SsCuAO1 are involved in stress tolerance in Sporisorium scitamineum. (a) Mating assay of wild‐type (WT) (MAT‐1 × MAT‐2), sspaoΔ (ss1paoΔ × ss2paoΔ) and sscuao1Δ (ss1cuao1Δ × ss2cuao1Δ) under treatment of NaCl (500 mM), sorbitol (1 M), Congo Red (CR, 1 mM) or SDS (0.1 mM), at 28°C for 2–3 days. Scale bar = 1 mm. (b) Mating cultures of WT (MAT‐1 × MAT‐2), sspaoΔ (ss1paoΔ × ss2paoΔ) or sscuao1Δ (ss1cuao1Δ × ss2cuao1Δ) were under H2O2 treatment (0.2 mM, 0.5 mM or 1 mM). The mating cultures were incubated at 28°C for 2–3 days before observation and imaging. Scale bar = 1 mm. (c) Measurement of intracellular H2O2 level in haploid sporidia of MAT‐1, ss1paoΔ and ss1cuao1Δ grown in liquid YePS medium for 2 days, using Amplex Red Hydrogen Peroxide/Peroxidase Assay kit. Bar chart depicts the mean ± SE derived from three independent biological repeats, each containing four replications (n = 4). (d) Transcriptional profile of catalase 1 (SsCAT1), catalase 2 (SsCAT2), peroxiredoxin 1 (SsPRX1), peroxidase (SsPOD), glutathione S‐transferase (SsGST), glutathione S‐transferase 1 (SsGST1), and superoxide dismutase 2 (SsSOD2) genes in MAT‐1, ss1paoΔ and ss1cuao1Δ. Bar chart depicts the mean ± SE derived from three independent biological repeats, each containing three replications (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, versus MAT‐1.

PAO‐ and CuAO‐mediated amine oxidation produce H2O2 as one of the products (Parsons et al., 1995; Walters, 2003); therefore, they may be potentially involved in tolerance to oxidative stress. We therefore evaluated tolerance to oxidative stress in sporidia of MAT‐1, ss1paoΔ or ss1cuao1Δ, grown on PDA containing various concentrations of H2O2. The ss1paoΔ and ss1cuao1Δ mutants showed no difference in sensitivity to H2O2 compared to MAT‐1 (Figure S5b). We also assessed the sexual mating/filamentation of S. scitamineum under oxidative stress. The result showed that a low concentration H2O2 (0.2 mM) slightly promoted filamentation of ss1paoΔ × ss2paoΔ. In contrast, ss1paoΔ × ss2paoΔ or ss1cuao1Δ × ss2cuao1Δ hardly produced hyphae under 1 mM H2O2, while MAT‐1 × MAT‐2 could still produce some extremely short hyphae (Figure 6b). We measured contents of H2O2 in MAT‐1, ss1paoΔ and ss1cuao1 and found that the intracellular H2O2 level was not significantly different in ss1paoΔ or ss1cuao1 compared to MAT‐1 (Figure 6c). Exogenous addition of antioxidants, including glutathione (GSH, 1 mM), N‐acetylcysteine (NAC, 1 mM), or vitamin C (VC, 1 mM), did not restore the defect of sexual mating/filamentation in ss1paoΔ × ss2paoΔ or ss1cuao1Δ × ss2cuao1Δ (Figure S5c). By RT‐qPCR, we assessed transcriptional levels of genes involved in redox homeostasis and/or response to oxidative stress, in MAT‐1, ss1paoΔ and ss1cuao1 mutants. The results showed that SsCAT1 and SsGST were significantly down‐regulated in ss1paoΔ, whereas SsCAT1, SsPRX1, SsPOD, and SsSOD2 were down‐regulated in ss1cuao1Δ, compared to MAT‐1 (Figure 6d).

We further assessed the role of SsPAO‐ and SsCuAO1‐mediated polyamine oxidation in modulating ROS accumulation during fungal infection to sugarcane. The results showed that exogenous added PUT promoted, while exogenous SPD inhibited, accumulation of ROS in sugarcane at the infection sites in the early stages (24–96 hpi; Figure 7a–c; Figure S6a). ROS accumulation was weaker in the sugarcane inoculated with sspaoΔ, sscuao1Δ, or ssatg8Δ mutant, at 48 hpi, but stronger than WT‐infected canes at 96 hpi (Figure 7d; Figure S6b). As a response to fungal infection, the RBOH (respiratory burst oxidase homologue) genes encoding ROS‐generating enzymes were up‐regulated in sugarcane inoculated with sspaoΔ or sscuao1Δ mutants compared to the WT‐infected sugarcane, at 96 hpi (Figure 7e), and might be responsible for the elevated ROS levels in the sspaoΔ or sscuao1Δ infected plants at this time point (Figure 7d; Figure S6b). On the other hand, the ROS scavenger encoding gene CAT1 was up‐regulated in sscuao1Δ‐infected sugarcane at 48 hpi but dropped to a comparable level to WT or sspaoΔ infected sugarcanes at 96 hpi (Figure 7e). These results indicate that changes in polyamine levels at the early stage of fungal infection affect redox homeostasis at the fungus–sugarcane interface, possibly by differentially regulating ROS generator genes in the infected plants. Therefore, SsPAO and SsCuAO1 function is required for modulating polyamine homeostasis at this stage.

FIGURE 7 SsPAO and SsCuAO1 affect host reactive oxygen species (ROS) accumulation during early infection stage. (a–d) Quantification of the amount of 3,3′‐diaminobenzidine (DAB) staining of the injected plant tissue based on the pixel intensity. Average optical density (AOD) = IOD (Integrated density)/Area, measured by ImageJ (v. 1.53 k). Bar chart depicts the mean ± SE derived from three independent biological repeats, each containing three replications (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, versus WT (Control). PUT, putrescine; SPD, spermidine; SPM, spermine. (e) Transcriptional profile of catalase 1 (CAT1, Gene ID: Sspon.01G0050630‐2D), peroxidase (POD, Gene ID: Sspon.02G0012680‐1A) and respiratory burst oxidase homologues (RBOH, Gene ID: Sspon.06G0014970‐3D) genes in sugarcane at 48 and 96 hours post‐inoculation (hpi). The sugarcane glyceraldehyde dehydrogenase (GAPDH) gene served as an internal control. Bar chart depicts the mean ± SE derived from three independent biological repeats, each containing three replications (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, versus WT (48 hpi).

Taken together, we conclude that SsPAO and SsCuAO1 are involved in stress tolerance and regulating host ROS accumulation in early infection stages of infection by S. scitamineum.

3 DISCUSSION

Our previous work proved that polyamines play differential roles in growth and pathogenic development of sugarcane smut fungus, probably by collaboratively regulating intracellular redox homeostasis with the cAMP‐PKA signalling pathway (Yin et al., 2024). In particular, a low level of PUT promoted, while a high level of PUT suppressed, filamentous growth after sexual mating (Yin et al., 2024). On the other hand, SPD plays a positive role in filamentous growth and pathogenicity (Yin et al., 2024). It has been reported that low concentrations of SPD significantly activate teliospore germination and polyamine levels increase in smut teliospores after contact with sugarcane glycoproteins as a plant‐defensive mechanism (Sanchez‐Elordi et al., 2019). In this study, we identified two potential polyamine oxidation enzymes, SsPAO and SsCuAO1, that also contribute to homeostasis of intracellular and extracellular polyamines. SsPAO and SsCuAO1 are both required for sporidia growth, sexual mating/filamentation, and pathogenicity.

Polyamine oxidation contributes to plant or animal cell development and/or death by modulating polyamine homeostasis and/or producing biologically active products, including H2O2 (Kang et al., 2023; Murray Stewart et al., 2018; Tavladoraki et al., 2016). A striking increase in maize polyamine biosynthesis, mainly free and conjugated PUT, occurrs in the tumours induced by the fungus and in the neighbouring plant tissues (Rodriguez‐Kessler et al., 2008). It has been reported that plant CuAOs mainly catalyse PUT and CAD oxidation, and PAOs oxidize SPD and SPM (Tavladoraki et al., 2016). rBjPAO1 and rBjPAO2 from amphioxus Branchiostoma japonicum were shown to catalyse oxidation of SPM and N 1‐acetyl‐SPM, respectively (Wang et al., 2016). The PAO gene was identified in the model fungal pathogen U. maydis and PAO was confirmed to catalyse conversion of SPD to PUT (Valdes‐Santiago et al., 2010). Saccharomyces cerevisiae Fms1 (yeast orthologue of PAO) can oxidize SPM, N 1‐acetyl‐SPM, N 1‐acetyl‐SPD, and N 8‐acetyl‐SPD, but not free SPD (Landry & Sternglanz, 2003). Compared to PAO, very little has been reported on CuAO enzymes in pathogenic fungi. Although CuAOs have been reported in Aspergillus spp. (Frebort et al., 1996, 1999; Sugawara et al., 2014, 2015), their oxidation substrates might not be polyamines. In this study, based on transcription changes of SsPAO or SsCuAO1 genes in response to treatment with different polyamines (Figure 1b), we infer that SsCuAO1 may be induced by SPM and SPD to catalyse oxidation of these two polyamines, while the oxidation product PUT suppresses SsCuAO1 transcription. On the other hand, SPD induced, while SPM suppressed, SsPAO transcription; therefore, we infer that SsPAO preferably catalyses oxidation of SPD (probably to produce PUT). It is unclear whether SsPAO or SsCuAO1 is involved in PUT oxidation, as exogenous PUT did not seem to induce transcription of SsPAO, and down‐regulated SsCuAO1 transcription (Figure 1b). However, loss of SsPAO or SsCuAO1 caused only accumulation of intracellular PUT but no obvious changes in SPD or SPM (Figure 1a). We infer that changes in polyamines may reflect the combined effect of disturbances of polyamine oxidation (direct effect) and polyamine biosynthesis (maybe indirectly). Given that loss of SsPAO or SsCuAO1 led to down‐regulation of SsSAMDC and SsSPDS/SPMS (both for converting PUT to SPD) (Figure 1c), we infer that accumulation of PUT in the ss1pao1Δ or ss1cuao1Δ mutants may be due to disruption of the PUT‐to‐SPD conversion, while the defect of SPD oxidation might be masked (or functionally redundant with other amide oxidases). Besides, exogenous addition of a high concentration (1 mM) of SPM affected yeast‐like growth of ss1cuao1Δ mutant, but not WT or ss1paoΔ mutant (Figure S3a,b), further supporting that SsCuAO1 may be involved in SPM oxidation, but SsPAO is not. Overall, we infer that the most likely substrate of SsPAO may be SPD, and the most likely substrates of SsCuAO1 may be SPD or SPM.

H2O2 is an important product of PAO‐ or CuAO‐mediated polyamine oxidation (Murray Stewart et al., 2018; Tavladoraki et al., 2016); therefore, PAOs and CuAOs are expected to play a role in regulating redox homeostasis under different physiological conditions. Plant PAOs exhibit the greatest diversity among eukaryotes, with origins linked to archaeal PAO‐like proteins and eukaryotic PAOs (Salvi & Tavladoraki, 2020). In the outer tissues of the maize mesocotyl, PAO is up‐regulated by light and down‐regulated by auxin (Cona et al., 2003). The transcriptional level of ZmPAO1 is up‐regulated and thus leads to increased H2O2 levels in maize tumours (Jasso‐Robles et al., 2016). GmPAO positively regulates plant resistance against the oomycetous pathogen Phytophthora sojae (Yang et al., 2022). The plant PAO activities were higher in the incompatible interaction at all the stages during Fusarium oxysporum f. sp. ricini–castor interaction (Mhaske et al., 2013). CuAO in Vicia faba guard cells is an essential enzymatic source for H2O2 production via degradation of PUT (An et al., 2008). CuAO1 of A. thaliana contributes to abscisic acid (ABA) and polyamines‐induced nitric oxide biosynthesis during stress response (Wimalasekera et al., 2011). Besides, osmotic stress could induce oxidation of polyamines to regulate defence responses and the susceptibility of grapevine to Botrytis cinerea (Hatmi et al., 2014). These studies demonstrate that plants modulate polyamine homeostasis by PAOs or CuAOs function, during which process H2O2 is produced and likely to act as a response to stressful conditions including pathogen infection. On the other hand, H2O2 production also contributes to fungal cell growth and development. It has been shown that the cAMP/PKA signalling pathway modulates expression of genes involved in redox homeostasis, thus regulating intracellular H2O2 accumulation that favours filamentous growth of S. scitamineum (Chang et al., 2019). However, this study showed that deletion of SsPAO or SsCuAO1 did not cause obvious changes in intracellular H2O2 levels (Figure 6c). Furthermore, exogenous addition of either H2O2 or antioxidants could not restore the filamentation defect of sspaoΔ or sscuao1Δ mutants (Figure 6b; Figure S5c), suggesting that SsPAO‐ or SsCuAO1‐mediated polyamine oxidation may not be a major contributor for generating intracellular H2O2. ROS‐generating and scavenging enzyme‐encoding genes, CAT1, POD, and RBOH, have been reported to be up‐regulated in sugarcane upon S. scitamineum infection (Wu et al., 2022, 2024). Our results showed that PUT promoted, while SPD inhibited, the accumulation of ROS, as quantified based on DAB staining with the infected sites of sugarcane stem at early infection stages (Figure 7a–c; Figure S6a), and such redox homeostasis during fungus–sugarcane interaction is probably regulated by SsPAO, SsCuAO1, and SsATG8 (Figure 7d; Figure S6b).

It has been reported that SPD induces autophagy in chondrocytes by inducing expression of the acetyltransferase EP300, which promotes autophagy activity, probably by post‐translational modification of autophagy proteins (Sacitharan et al., 2018). Similarly, SPD can induce autophagy in mice and human cells through post‐translational modification of the translation factor EIF5A, which in turn increases the synthesis of the master transcriptional factor TFEB that governs autophagy (Zhang & Simon, 2020). In cucumber, SPD induces expression of autophagy and the RBOH gene, both contributing to autophagy induction (Zhang et al., 2021). SPM‐induced autophagy in plants is accompanied by an increase in the secondary messengers ROS and nitric oxide (NO) (Dmitrieva et al., 2018). From these reports, we can see that polyamines could promote autophagy by directing activating expression of autophagy genes, or in an indirect way by promoting ROS (including H2O2) levels. On the other hand, the cAMP/PKA pathway has been shown to be involved in regulating autophagy activity in yeast and fungal cells (Perez‐Diaz et al., 2023; Stephan et al., 2010; Yu & Rollins, 2022). In this study, we showed that individual addition of cAMP, PUT, SPD, or SPM could induce autophagy in S. scitamineum (Figure 5a,b). We further confirmed that autophagy activity was enhanced in the ss1paoΔ mutant and reduced in ss1cuao1Δ (Figure 5c,d). SsATG8 was up‐regulated in the ss1paoΔ mutant after a 4 h exposure to nitrogen starvation (MM−N), but there was no significant difference in ss1cuao1Δ under MM−N compared to YePS medium (Figure S4d). We infer that polyamine‐based regulation of autophagy is through expression of autophagy genes, which may also dependent on the cAMP/PKA pathway. Furthermore, cAMP/polyamine‐based regulation of autophagy may contribute to S. scitamineum filamentation and/or pathogenicity. Treatment with rapamycin, a chemical targeting TOR kinase to induce autophagy (Wang & Zhang, 2019), slightly enhanced filamentation of the ss1cuao1Δ mutant while slightly suppressing filamentation of the ss1paoΔ mutant (Figure S4e). Our previous study shows that pseudohyphae increase in ssatg8Δ mutant, which may account for loss of pathogenicity in this mutant (Zhang et al., 2019). We infer that dysregulated autophagy in the sspaoΔ or sscuao1Δ mutants may also be responsible for loss or reduced pathogenicity.

In summary, our current and previous (Yin et al., 2024) works reveal that biosynthesis and oxidation of polyamines serve a mechanism for maintaining intracellular homeostasis of polyamines and ROS (H2O2), interconnecting with the cAMP‐PKA signalling pathway and autophagy for contribution to S. scitamineum filamentous growth and pathogenicity. A proposed working model is illustrated in Figure 8.

FIGURE 8 A proposed working model. Polyamine biosynthesis and oxidation together maintain intracellular homeostasis of polyamines, especially putrescine (PUT) and spermidine (SPD), which are capable of regulating intracellular reactive oxygen species (ROS) (H2O2) and autophagy, thereby contributing to the filamentous growth and pathogenicity of Sporisorium scitamineum.

4 EXPERIMENTAL PROCEDURES

4.1 Fungal strains and growth conditions

The wild‐type strains of MAT‐1 (a1 b1) and MAT‐2 (a2 b2), previously isolated, identified, and maintained by our laboratory, were used for the generation of all transformations/mutants in this study (Yan et al., 2016). Details of the fungal strains generated and used in this study are listed in Table S2. The culture media employed include yeast extract‐peptone‐sucrose medium (YePS; yeast extract 1%, peptone 2%, sugar 2%, pH 6.5), minimal medium (MM; yeast nitrogen base 0.17%, glucose 2%, 10 mM ammonium sulphate, pH 6.8), and potato dextrose agar (PDA powder 4%; DingGuo).

For sexual mating assay, fresh haploid sporidia were cultured in liquid YePS medium overnight. Subsequently, the sporidia were collected after washing twice with sterile double‐distilled water and the concentration was adjusted to OD600 = 1.0. The haploid sporidia of opposite mating types were mixed at equal volume (OD600 = 1.0) and spotted on PDA or solid MM, and kept in the dark in a 28°C incubator for 2–3 days before photographing.

For haploid growth analysis, fresh haploid sporidia were cultured in liquid YePS medium overnight, washed twice with sterile double‐distilled water, and adjusted to OD600 = 1.0. Fresh haploid sporidia (OD600 = 1.0, equivalent to cfu = 107/mL) of WT and mutants were diluted in a gradient into 107, 106, 105, 104, and 103 spotted onto PDA or MM, and kept in dark in a 28°C incubator for 2–3 days before photographing.

For growth curve analysis, fresh haploid sporidia were cultured in liquid YePS medium overnight, washed twice with fresh liquid YePS medium, and adjusted to OD600 = 0.01, then added to 120 μL of the culture into a 100‐well honeycomb plate, incubated in the automatic microbial growth curve analyser (Oy Growth Curves Ab Ltd; Bioscreen C) with the setting as 28°C, for 3 days.

4.2 Nucleic acid‐related manipulation

Genomic DNA of S. scitamineum strains was extracted from the sporidia culture grown on solid YePS medium for 2 days at 28°C, using a Fungal DNA Midi Kit (Omega). Southern blot analysis was performed with the digoxigenin (DIG) High Prime DNA labelling and detection starter kit II (Roche). For verification of ss1paoΔ and ss2paoΔ mutants, 5 μg genomic DNA of indicated strains was digested with HindIII/PstI (NEB). For verification of ss1cuao1Δ and ss2cuao1Δ mutants, 5 μg genomic DNA of indicated strains was digested with PvuI/SacI (NEB). Digested products were electrophoresed in a 0.75% agarose gel and then transferred onto Hybond‐N+ membrane (Amersham). The specific probe labelled with DIG‐11‐dUTP using DIG‐High prime was PCR amplified from the 5′ flanking sequence of the SsPAO gene and the 3′ flanking sequence of the SsCuAO1 gene. Hybridization and detection were performed according to the instruction manual provided by Roche Applied Science.

Total RNA extract from fresh haploid sporidia was performed with FastPure Plant Total RNA Isolation Kit (Vazyme) following the kit instructions. The mating culture of WT or mutants was mixed with the compatible sporidia and inoculated on solid YePS medium at 28°C for 2 days, before total RNA extraction. PUT, SPD, or SPM treatment (1 mM) was carried out 90 min before total RNA extraction, no chemicals were added as a control. For cDNA synthesis, total RNA from each strain was reverse transcribed using the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper). qPCR with cDNA as template was run on the QuantStudio 6 Flex (Life Technologies) with ChamQ Universal SYBR qPCR Master Mix (Vazyme). Relative gene expression level was calculated with −ΔΔCt method (Livak & Schmittgen, 2001) with the cytoskeletal protein gene ACTIN as an internal control. The experiment was conducted in triplicate and for two independent biological replicates.

4.3 Plasmid construction and fungal transformation

Double‐joint PCR was performed to construct the fragment for the replacement of resident target gene by the hygromycin (HPT) or zeocin (ZEO) gene following the strategy described previously (Yu et al., 2004). Polyethylene glycol (PEG)‐mediated protoplast transformation was used to delete and complement the target gene by following the established protocols (Chang et al., 2019; Cui et al., 2022). For generation of deletion mutants, the flanking DNA (1 kb 5′ and 3′) of the resident target gene was PCR amplified using genomic DNA of MAT‐1 strain as the template and the HPT gene with plasmid pEX2 as the template using the primer pairs (Table S3). For generation of SsCuAO1 complemented strains, the encoding sequences were PCR amplified using the primer pairs (Table S3). The PCR products were ligated into the vector pEX2‐zeocin‐dsRed to create an in‐frame fusion with the RFP‐encoding sequence at its C‐terminus, driven by the original promoter. The recombinant plasmid was sequenced for verification, before being transferred into the corresponding mutant strains by PEG‐mediated protoplast transformation. For generation of SsPAO complementation strains, a reintegrated homologous fragment, composed of native promoter of SsPAO (LB‐SsPAO), coding sequence of GFP and SsPAO, expression cassette of ZEO R gene, and the downstream fragment of SsPAO‐RB, was amplified by fusion PCR, and then transformed into ss1pao∆ or ss2pao∆ protoplasts through PEG‐mediated protoplast transformation. The complementation strains were verified by PCR amplification using the primers listed in Table S3. For generation of SsPRF1‐overexpressing strains, a reintegrated homologous fragment, composed of the upstream fragment of LB‐SsPRF1, the strong constitutive promoter P GAPD , coding sequence of SsPRF1, expression cassette of ZEO R gene, and the downstream fragment of SsPRF1‐RB, was amplified by fusion PCR, and then transformed into ss1pao∆ or ss1cuao1∆ protoplasts through PEG‐mediated protoplast transformation. The overexpressing strains were verified by PCR amplification using the primers listed in Table S3. For generation of GFP‐SsATG8:MAT‐1 strain, a reintegrated homologous fragment, composed of the native promoter of SsATG8 (LB‐SsATG8), coding sequence of GFP and SsATG8, expression cassette of ZEO R gene, and the downstream fragment of SsATG8‐RB, was amplified by fusion PCR, and then transformed into MAT‐1 protoplasts through PEG‐mediated protoplast transformation. All hygromycin‐resistant or zeocin‐resistant transformants were firstly screened by PCR and then confirmed by Southern blot analysis or fluorescence detection.

4.4 Chemical treatment

Putrescine (PUT; Sigma‐Aldrich), spermidine trihydrochloride (SPD; Sigma‐Aldrich), spermine (SPM; Sigma‐Aldrich), adenosine 3′,5′‐cyclic monophosphate (3′,5′‐cAMP; Sigma‐Aldrich), H2O2 (DaMao), N‐acetyl‐L‐cysteine (NAC; Aladdin), GSH (glutathione reduced; Aladdin), Vitamin C (VC; Aladdin), and rapamycin (RAPA; Aladdin) were used in this study.

4.5 Relative fungal biomass and pathogenicity assays

Fungal biomass assay of the inoculated sugarcane seedlings was carried out according to our previously described (Yin et al., 2024). The haploid sporidia of S. scitamineum strains were grown on liquid YePS medium in a shaking incubator at 28°C for 1–2 days. The fresh haploid sporidia were collected, resuspended in the sterile double‐distilled water, adjusted to 107 cells/mL, and mixed with haploid sporidia of opposite mating types in equal volume (1:1) and 1 mL of such mixture was injected into sugarcane seedlings, and total DNA of the inoculated sugarcane tissue was extracted at 3 and 7 dpi. The relative fungal biomass was measured using the fungal ACTIN gene as a reference, whereas the sugarcane glyceraldehyde dehydrogenase (GAPDH) gene served as an internal control.

For assessment of pathogenicity, sugarcane stems of susceptible cultivar ROC22 were soaked in 1 L mixture (1:1 vol/vol) of MAT‐1 × MAT‐2, ss1paoΔ × ss2paoΔ or ss1cuao1Δ × ss2cuao1Δ sporidia (107 cells/mL for each), at 28°C for 30 min, and then planted in pots (five or six seedlings per pot). Two weeks later, the seedlings were further inoculated with 1 mL mixture (1:1 vol/vol) of MAT‐1 × MAT‐2, ss1paoΔ × ss2paoΔ and ss1cuao1Δ × ss2cuao1Δ. Wild‐type mixture MAT‐1 × MAT‐2 served as a positive control and double‐distilled water inoculation as a negative control. Inoculated plants were kept in a greenhouse with the setting as 28°C, 80% humidity, and a 12 h/12 h light/dark cycle, for 3–6 months. Pathogenicity assay for the wild‐type and all mutants was repeated two times and each replicate with at least 20 plants. The symptoms of black whip were documented and photographed at about 6 months post‐inoculation. Meanwhile, the plant height of sugarcane was measured. Percentage (%) of black whip/total seedlings was estimated.

4.6 Microscopic observation and imaging

Fresh haploid sporidia were cultured in liquid YePS medium at 28°C, 200 rpm overnight. The GFP or RFP signals were observed and photographed in S. scitamineum haploid sporidia, using an Axio Observer Z1 microscope (Zeiss) equipped with sCMOS camera (PCO Edge).

4.7 Histochemical localization of H2O2

The histochemical localization of H2O2 following the established methods with some modification (Libik‐Konieczny et al., 2015). Sugarcane leaves were infiltrated with a solution of 2 mg/mL 3,3′‐diaminobenzidine (DAB; Macklin) prepared in water, pH 5.5. Infiltration was carried for 1 h in the dark at room temperature. DAB forms a deep‐brown polymerization product upon reaction with H2O2 in the presence of peroxidases. Samples incubated in DAB were rinsed with distilled water. Chlorophyll was extracted with ethanol:chloroform (80:20; vol/vol) supplemented with 0.15% (vol/vol) trichloroacetic acid. The bleached leaves were submerged in a glycerol:water (1:1 vol/vol) solution and pictures were taken using an Axio Observer Z1 microscope equipped with sCMOS camera.

4.8 Quantification of polyamines, H2O2 , and cAMP

The polyamines were extracted and detected following the established methods with some modifications (Yin et al., 2024; Zeljkovic et al., 2024). Frozen cells were homogenized in 4 mL of 5% (wt/vol) with cold perchloric acid solution, and the homogenate was kept in ice for 60 min. The mixture was then centrifuged at 14,000 g at 4°C for 30 min. Then, 500 μL of supernatant was transferred to a 10 mL centrifuge tube and the supernatant sample derivatized using benzoyl chloride. The sample solution was analysed using the Thermo Scientific UltiMate 3000 Rapid Separation Quaternary System Liquid Mass Spectrometry (LC–MS) using Single Ion Monitoring (SIM) single ion detection scan mode, reversed‐phase column C18 (Supelco Discovery, 25 cm × 4.6 mm, particle size 5 μm), column temperature 40°C, flow rate 0.2 mL/min, injection volume 10 μL aliquots. Benzoylated polyamines were eluted with 60% (vol/vol) methanol. Signal mass spectrometry scans were set to PUT at 297.16 m/z, SPD at 458.24 m/z, and SPM at 619.33 m/z. Data analysis was performed using the Thermo Xcalibur software (Thermo Fisher Scientific).

Intracellular H2O2 was quantified by a Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (ThermoFisher) and following the manufacturer's instruction. The fresh haploid sporidia were collected after cultured in liquid YePS medium for 2 days at 28°C and fungal samples were then collected by centrifugation. After grinding the fungus with liquid nitrogen, 0.1 g of fungal sample was dissolved in 10 times the volume of 1 × buffer, and the supernatant was obtained by centrifugation for subsequent analysis.

The fresh haploid sporidia were collected after growing on liquid YePS medium for 16 h at 28°C before cAMP extraction and detection following a previously described method (Chang et al., 2019). The dikaryotic hyphae were collected after growing on solid YePS medium for 2 days and then intracellular cAMP was extracted and quantified.

4.9 Immunoblotting

To monitor the autophagic process, the GFP‐SsATG8 fusion construct driven by the original promoter and a full‐length SsATG8 gene coding region was transformed into MAT‐1. The GFP‐SsATG8:MAT‐1, MAT‐1, ss1paoΔ and ss1cuao1Δ strains were first cultured in liquid YePS medium at 28°C for 16 h, and then washed with sterile double‐distilled water and subjected to nitrogen starvation (cultured in MM−N for 4 h) to induce non‐selective autophagy. For immunoblot analysis, the total protein was extracted from the fresh haploid sporidia after inducing non‐selective autophagy. Protein samples were fractioned by SDS‐PAGE gels (Smart‐Lifesciences, 4%–12%, for detection using anti‐GFP antibody, or SurePAGE, Bis‐Tris, 15%, for detection using anti‐ATG8 antibody), then transferred to a polyvinylidene difluoride (PVDF) membrane. Immunoblotting for GFP‐ATG8 cleavage was performed with primary antibody (anti‐GFP antibody; Abcam) at the recommended dilutions. Immunoblotting for ATG8 cleavage was performed with primary antibody (anti‐ATG8 antibody; MBL). Horseradish peroxidase (HRP)‐conjugated goat anti‐mouse IgG (Abbkine) was used as the secondary antibody. The efficient chemiluminescence kit (Genview) was used to detect the chemiluminescent signals. The intensity of the protein bands was determined by the ImageJ software.

4.10 Statistical analysis

Data were presented as mean ± SE. Statistical analysis was performed using GraphPad Prism 7 software, and p < 0.05 were considered statistically significant.

CONFLICT OF INTEREST STATEMENT

No conflict of interest exists during the conduct of this research and in preparation of this manuscript.

Supporting information

Figure S1.

Figure S2.

Figure S3.

Figure S4.

Figure S5.

Figure S6.

Table S1.

Table S2.

Table S3.

ACKNOWLEDGEMENTS

This work was supported by National Natural Science Foundation of China (32102156 and U23A20148). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

DATA AVAILABILITY STATEMENT

The authors confirm that the data supporting the findings of this study are available within the article and/or its supplementary materials.
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