Polyamine oxidation enzymes regulate sexual mating/filamentation and pathogenicity in Sporisorium scitamineum.

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.

Fungal Diversity and Gibberellin Hormones Associated with Long Whips of Smut-Infected Sugarcanes.

Sugarcane smut, caused by the fungus Sporisorium scitamineum (Sydow), significantly affects sugarcane crops worldwide. Infected plants develop whip-like structures known as sori. Significant variations in these whip lengths are commonly observed, but the physiological and molecular differences causing these morphological differences remain poorly documented. To address this, we employed conventional microbe isolation, metagenomic, and metabolomic techniques to investigate smut-infected sugarcane stems and whips of varying lengths. Metagenomics analysis revealed a diverse fungal community in the sugarcane whips, with Sporisorium and Fusarium genera notably present (>1%) in long whips. Isolation techniques confirmed these findings. Ultra-performance liquid chromatography analysis (UHPLC-MS/MS) showed high levels of gibberellin hormones (GA3, GA1, GA4, GA8, and GA7) in long whips, with GA4 and GA7 found exclusively in long whips and stems. Among the prominent genera present within long whips, Fusarium was solely positively correlated with these gibberellin (GA) hormones, with the exception of GA8, which was positively correlated with Sporisorium. KEGG enrichment analysis linked these hormones to pathways like diterpenoid biosynthesis and plant hormone signal transduction. These findings suggest that Fusarium may influence GA production leading to whip elongation. Our study reveals fungal dynamics and gibberellin responses in sugarcane smut whips. Future research will explore the related molecular gibberellin synthesis mechanisms.

SsCyp86 modulates sporisorium scitamineum mating/filamentation and pathogenicity through regulating fatty acid metabolism.

BACKGROUND: Fatty acid metabolism constitutes a significant and intricate biochemical process within microorganisms. Cytochrome P450 (CYP450) enzymes are found in most organisms and occupy a pivotal position in the metabolism of fatty acids. However, the role of CYP450 enzyme mediated fatty acid metabolism in the pathogenicity of pathogenic fungi remains unclear. METHODS: In this study, a CYP450 enzyme-encoding gene, SsCYP86, was identified in the sugarcane smut fungus Sporisorium scitamineum and its functions were characterized using a target gene homologous recombination strategy and metabonomics. RESULTS: We found that the expression of SsCYP86 was induced by or sugarcane wax or under the condition of mating/filamentation. Sexual reproduction assay demonstrated that the SsCYP86 deletion mutant was defective in mating/filamentation and significantly reduced its pathogenicity. Further fatty acid metabolomic analysis unravelled the levels of fatty acid metabolites were reduced in the SsCYP86 deletion mutant. Exogenous addition of fatty acid metabolites cis-11-eicosenoic acid (C20:1N9), pentadecanoic acid (C15:0), and linolenic acid (C18:3N3) partially restored the mating/filamentation ability of the SsCYP86 deletion mutant and restored the transcriptional level of the SsPRF1, a pheromone response transcription factor that is typically down-regulated in the absence of SsCYP86. Moreover, the constitutive expression of SsPRF1 in the SsCYP86 deletion mutant restored its mating/filamentation. CONCLUSION: Our results indicated that SsCyp86 modulates the SsPRF1 transcription by fatty acid metabolism, and thereby regulate the sexual reproduction of S. scitamineum. These findings provide insights into how CYPs regulate sexual reproduction in S. scitamineum.

Transcriptional Regulation of SugarCane Response to Sporisorium scitamineum: Insights from Time-Course Gene Coexpression and Ca2+ Signaling.

Sugarcane response to Sporisorium scitamineum is determined by multiple major genes and numerous microeffector genes. Here, time-ordered gene coexpression networks were applied to explore the interaction between sugarcane and S. scitamineum. Totally, 2459 differentially expressed genes were identified and divided into 10 levels, and several stress-related subnetworks were established. Interestingly, the Ca2+ signaling pathway was activated to establish the response to sugarcane smut disease. Accordingly, two CAX genes (ScCAX2 and ScCAX3) were cloned and characterized from sugarcane. They were significantly upregulated under ABA stress but inhibited by MeJA treatment. Furthermore, overexpression of ScCAX2 and ScCAX3 enhanced the susceptibility of transgenic plants to the pathogen infection, suggesting its negative role in disease resistance. A regulatory model for ScCAX genes in disease response was thus depicted. This work helps to clarify the transcriptional regulation of sugarcane response to S. scitamineum stress and the function of the CAX gene in disease response.

An efficient inoculation method to evaluate virulence differentiation of field strains of sugarcane smut fungus.

Sugarcane smut, caused by the fungal pathogen Sporisorium scitamineum, is a prominent threat to the sugarcane industry. The development of smut resistant varieties is the ultimate solution for controlling this disease, due to the lack of other efficient control methods. Artificial inoculation method is used to evaluate the virulence differentiation of pathogens. The mostly used artificial inoculation methods are soaking of the seed canes in the teliospore solution and injection of teliospores or haploid sporidia into the sugarcane sprouts. However, due to the infection nature of the pathogen that invades the sugarcane plant through meristem tissue of the sprout or shoot, the rate of successful infection is often low and fluctuated, resulting in low confidence of the assays. We recently reported a rapid and high-throughput inoculation method called plantlet soaking by using tissue culture-derived sugarcane plantlets as the test plants. Here, we compare different inoculation methods and report the characterization of parameters that may affect the sensitivity and efficiency of the plantlet soaking technique. The results showed that sugarcane plantlets were highly vulnerable to infection, even with the inoculum density at 6.0 × 105 basidial spores/ml, and this method could be applied to all varieties tested. Notably, varieties showing high smut resistance in the field exhibited high susceptibility when inoculated with the plantlet soaking method, suggesting that the plantlet soaking method is a good complement to the traditional methods for screening germplasms with internal resistance. In addition, this method could also be used to monitor the variation of cellular virulence of the smut pathogen strains in the field.

Discovery of N-Benzyl-4-(1-bromonaphthalen-2-yl)oxybutan-1-amine as a Potential Antifungal Agent against Sporidia Growth and Teliospore Germination of Sporisorium scitamineum.

The cultivation of sugar cane using perennial roots is the primary planting method, which is one of the reasons for the serious occurrence of sugar cane smut disease caused by the basidiomycetous fungus Sporisorium scitamineum in the sugar cane perennial root planting area. Consequently, it is crucial to eliminate pathogens from perennial sugar cane buds. In this study, we found that MAP kinase Hog1 is necessary for heat stress resistance. Subsequent investigations revealed a significant reduction in the expression of the heat shock protein 104-encoding gene, SsHSP104, in the ss1hog1Δ mutant. Additionally, the overexpression of SsHSP104 partially restored colony growth in the ss1hog1Δ strain following heat stress treatment, demonstrating the crucial role of SsHsp104 in SsHog1-mediated heat stress tolerance. Hence, we constructed the ss1hsp104:eGFP fusion strain in the wild type of S. scitamineum to identify small-molecule compounds that could inhibit the heat stress response, leading to the discovery of N-benzyl-4-(1-bromonaphthalen-2-yl)oxybutan-1-amine as a potential compound that targets the SsHog1 mediation SsHsp104 pathway during heat treatment. Furthermore, the combination of N-benzyl-4-(1-bromonaphthalen-2-yl)oxybutan-1-amine and warm water treatment (45 °C for 15 min) inhibits the growth of S. scitamineum and teliospore germination, thereby reducing the occurrence of sugar cane smut diseases and indicating its potential for eliminating pathogens from perennial sugar cane buds. In conclusion, these findings suggest that N-benzyl-4-(1-bromonaphthalen-2-yl)oxybutan-1-amine is promising as a targeted compound for the SsHog1-mediated SsHsp104 pathway and may enable the reduction of hot water treatment duration and/or temperature, thereby limiting the occurrence of sugar cane smut diseases caused by S. scitamineum.

Intracellular polyamines regulate redox homeostasis with cAMP-PKA signalling during sexual mating/filamentation and pathogenicity of Sporisorium scitamineum.

Sugarcane smut caused by Sporisorium scitamineum seriously impairs sugarcane production and quality. Sexual mating/filamentation is a critical step of S. scitamineum pathogenesis, yet the regulatory mechanisms are not fully understood. In this study, we identified the SsAGA, SsODC, and SsSAMDC genes, which are involved in polyamine biosynthesis in S. scitamineum. Deletion of SsODC led to complete loss of filamentous growth after sexual mating, and deletion of SsAGA or SsSAMDC caused reduced filamentation. Double deletion of SsODC and SsSAMDC resulted in auxotrophy for putrescine (PUT) and spermidine (SPD) when grown on minimal medium (MM), indicating that these two genes encode enzymes that are critical for PUT and SPD biosynthesis. We further showed that low PUT concentrations promoted S. scitamineum filamentation, while high PUT concentrations suppressed filamentation. Disrupted fungal polyamine biosynthesis also resulted in a loss of pathogenicity and reduced fungal biomass within infected plants at the early infection stage. SPD formed a gradient from the diseased part to nonsymptom parts of the cane stem, suggesting that SPD is probably favourable for fungal virulence. Mutants of the cAMP-PKA (SsGPA3-SsUAC1-SsADR1) signalling pathway displayed up-regulation of the SsODC gene and elevated intracellular levels of PUT. SsODC directly interacted with SsGPA3, and sporidia of the ss1uac1ΔodcΔ mutant displayed abundant pseudohyphae. Furthermore, we found that elevated PUT levels caused accumulation of intracellular reactive oxygen species (ROS), probably by suppressing transcription of ROS-scavenging enzymes, while SPD played the opposite role. Overall, our work proves that polyamines play important roles in the pathogenic development of sugarcane smut fungus, probably by collaboratively regulating intracellular redox homeostasis with the cAMP-PKA signalling pathway.

Cytochrome c-peroxidase modulates ROS homeostasis to regulate the sexual mating of Sporisorium scitamineum.

IMPORTANCE: Reactive oxygen species play an important role in pathogen-plant interactions. In fungi, cytochrome c-peroxidase maintains intracellular ROS homeostasis by utilizing H2O2 as an electron acceptor to oxidize ferrocytochrome c, thereby contributing to disease pathogenesis. In this study, our investigation reveals that the cytochrome c-peroxidase encoding gene, SsCCP1, not only plays a key role in resisting H2O2 toxicity but is also essential for the mating/filamentation and pathogenicity of S. scitamineum. We further uncover that SsCcp1 mediates the expression of SsPrf1 by maintaining intracellular ROS homeostasis to regulate S. scitamineum mating/filamentation. Our findings provide novel insights into how cytochrome c-peroxidase regulates sexual reproduction in phytopathogenic fungi, presenting a theoretical foundation for designing new disease control strategies.

A Genetically Engineered Escherichia coli for Potential Utilization in Fungal Smut Disease Control.

Sporisorium scitamineum, the basidiomycetous fungus that causes sugarcane smut and leads to severe losses in sugarcane quantity and quality, undergoes sexual mating to form dikaryotic hyphae capable of invading the host cane. Therefore, suppressing dikaryotic hyphae formation would potentially be an effective way to prevent host infection by the smut fungus, and the following disease symptom developments. The phytohormone methyl jasmonate (MeJA) has been shown to induce plant defenses against insects and microbial pathogens. In this study, we will verify that the exogenous addition of MeJA-suppressed dikaryotic hyphae formation in S. scitamineum and Ustilago maydis under in vitro culture conditions, and the maize smut symptom caused by U. maydis, could be effectively suppressed by MeJA in a pot experiment. We constructed an Escherichia coli-expressing plant JMT gene, encoding a jasmonic acid carboxyl methyl transferase that catalyzes conversion from jasmonic acid (JA) to MeJA. By GC-MS, we will confirm that the transformed E. coli, designated as the pJMT strain, was able to produce MeJA in the presence of JA and S-adenosyl-L-methionine (SAM as methyl donor). Furthermore, the pJMT strain was able to suppress S. scitamineum filamentous growth under in vitro culture conditions. It waits to further optimize JMT expression under field conditions in order to utilize the pJMT strain as a biocontrol agent (BCA) of sugarcane smut disease. Overall, our study provides a potentially novel method for controlling crop fungal diseases by boosting phytohormone biosynthesis.

Research progress on microbial control of sugarcane smut.

Sugarcane is the most important sugar crop. Sugarcane smut is one of the major diseases, which could reduce sugarcane yield and quality and seriously threaten the sustainable and healthy development of sugar industry. Microbial control of sugarcane smut is a rapidly emerging green biocontrol technology, with advantage to increase environmental compatibility and soil fertility. In this review, we briefly described the characteristics of Sporisorium scitamineum which causes sugarcane smut, synthesized the the mechanisms underlying the infection of sugarcane by S. scitamineum, and presented the research status of microbial controls of sugarcane smut via the application of bio-organic fertilizers and biopesticides. We then reviewed the mechanisms underlying the suppression of sugarcane smut by microorganisms through competition with pathogens for nutrients and ecological niches, secreting antagonistic substances, and improving plant resistance. It is notable that there are still some problems in the application of microbial control technologies, including poor colonization ability and unstable biocontrol efficiency. Finally, the major directions of future research on the biocontrol of sugarcane smut were proposed from the perspective of improving the biocontrol efficiency. This review would benefit the microbial control of sugarcane smut and the healthy development of sugar industry.

Enhanced Resistance to Fungal and Bacterial Diseases Due to Overexpression of BSR1, a Rice RLCK, in Sugarcane, Tomato, and Torenia.

Sugarcane smut caused by Sporisorium scitamineum is one of the most devastating sugarcane diseases. Furthermore, Rhizoctonia solani causes severe diseases in various crops including rice, tomato, potato, sugar beet, tobacco, and torenia. However, effective disease-resistant genes against these pathogens have not been identified in target crops. Therefore, the transgenic approach can be used since conventional cross-breeding is not applicable. Herein, the overexpression of BROAD-SPECTRUM RESISTANCE 1 (BSR1), a rice receptor-like cytoplasmic kinase, was conducted in sugarcane, tomato and torenia. BSR1-overexpressing tomatoes exhibited resistance to the bacteria Pseudomonas syringae pv. tomato DC3000 and the fungus R. solani, whereas BSR1-overexpressing torenia showed resistance to R. solani in the growth room. Additionally, BSR1 overexpression conferred resistance to sugarcane smut in the greenhouse. These three BSR1-overexpressing crops exhibited normal growth and morphologies except in the case of exceedingly high levels of overexpression. These results indicate that BSR1 overexpression is a simple and effective tool for conferring broad-spectrum disease resistance to many crops.

Kynurenine 3-Monooxygenase Gene SsCI51640 Is Required for Sporisorium scitamineum Mating/Filamentation by Regulating cAMP Pathway and Improving Sporidia Environmental Adaptability.

Sugarcane smut is a serious disease caused by Sporisorium scitamineum, which causes significant losses to the sugar industry. It is critical to reveal the molecular pathogenic mechanism of S. scitamineum to explore a new control strategy for sugarcane smut. On the basis of transcriptome sequencing data of two S. scitamineum strains with different pathogenicity, we identified the gene, SsCI51640, which was predicted to encode kynurenine 3-monooxygenase. In this study, we obtained knockout mutants and complementary mutants of this gene and identified gene function. The results showed that the sporidial growth rate and acid production ability of knockout mutants were significantly higher and stronger than those of the wild-type and complementary mutants. The growth of knockout mutants under abiotic stress (osmotic stress and cell wall stress) was significantly inhibited. In addition, the sexual mating ability and pathogenicity of knockout mutants were significantly reduced, while this phenomenon could be restored by adding exogenous cyclic adenosine monophosphate (cAMP). It is thus speculated that the SsCI51640 gene may regulate sexual mating and pathogenicity of S. scitamineum by the cAMP signaling pathway. Moreover, the SsCI51640 gene enhanced the sporidial environmental adaptability, which promoted sexual mating and development of pathogenicity. This study provides a theoretical basis for the molecular pathogenesis of S. scitamineum.

Assessment of the rhizosphere fungi and bacteria recruited by sugarcane during smut invasion.

Whip smut is one of the most serious and widely spread sugarcane diseases. Plant-associated microbes play various roles in conferring advantages to the host plant. Understanding the microbes associated with sugarcane roots will help develop strategies for the biocontrol of smut. Therefore, the present study explored microbe-mediated sugarcane response to smut invasion via 16S rRNA and ITS metabarcoding survey of the rhizosphere soils of resistant and susceptible sugarcane varieties. The bacterial and fungal diversity in the rhizosphere soils differed between the resistant and susceptible varieties. The bacterial genera Sphingomonas, Microcoleus_Es-Yyy1400, Marmoricola, Reyranella, Promicromonospora, Iamia, Phenylobacterium, Aridibacter, Actinophytocola, and Edaphobacter and one fungal genus Cyphellophora were found associated with smut resistance in sugarcane. Detailed analysis revealed that the majority of bacteria were beneficial, including the actinomycete Marmoricola and Iamia and Reyranella with denitrification activity. Analysis of bacterial network interaction showed that three major groups interacted during smut invasion. Meanwhile, seven of these genera appeared to interact and promote each other's growth. Finally, functional annotation based on the Functional Annotation of Prokaryotic Taxa (FAPROTAX) database predicted that the abundant bacteria are dominated by oxygenic photoautotrophy, photoautotrophy, and phototrophy functions, which may be related to smut resistance in sugarcane. The present study thus provides new insights into the dynamics of the sugarcane rhizosphere microbial community during smut invasion.

SsUbc2, a determinant of pathogenicity, functions as a key coordinator controlling global transcriptomic reprogramming during mating in sugarcane smut fungus.

The basidiomycete fungus Sporisorium scitamineum is the causative agent of sugarcane smut disease. Mating between two strains of the opposite mating type is essential for filamentous growth and infection in sugarcane plants. However, the mechanisms underlying mating and pathogenicity are still not well understood. In this work we used gene disruption to investigate the role of Ssubc2, the gene encoding a kinase regulator in S. scitamineum. Deletion of Ssubc2 did not alter the haploid cell morphology or growth rate in vitro or tolerance to stress, but mutants with both alleles deleted lost mating ability and infectivity. Deletion of one Ssubc2 allele in a pair with a wild-type strain resulted in impaired mating and reduced virulence. Transcriptome profiling revealed that about a third of genes underwent reprogramming in the wild types during mating. Although gene expression reprogramming occurred in the pairing of Ssubc2-null mutants, their transcriptomic profile differed significantly from that of the wild types, in which 625 genes differed from those present in the wild types that seemed to be among the required genes for a successful mating. These genes include those known to regulate mating and pathogenicity, such as components of the MAPK pathway and hgl1. Additionally, a total of 908 genes were differentially expressed in an out-of-control manner in the mutants. We conclude that SsUbc2 functions as a key factor to coordinate the reprogramming of gene expression at the global level and is essential for the transition from monokaryotic basidial growth to dikaryotic hyphal growth through mating.

Identification of Gene Modules and Hub Genes Associated with Sporisorium scitamineum Infection Using Weighted Gene Co-Expression Network Analysis.

Sporisorium scitamineum is a biotrophic fungus responsible for sugarcane smut disease. To investigate the key genes involved in S. scitamineum infection, we conducted RNA sequencing of sugarcane sprouts inoculated with S. scitamineum teliospores. A weighted gene co-expression network analysis (WGCNA) showed that two co-expressed gene modules, MEdarkturquoise and MEpurple-containing 66 and 208 genes, respectively-were associated with S. scitamineum infection. The genes in these two modules were further studied using Gene Ontology (GO) enrichment analysis, pathogen-host interaction (PHI) database BLASTp, and small secreted cysteine-rich proteins (SCRPs) prediction. The top ten hub genes in each module were identified using the Cytohubba plugin. The GO enrichment analysis found that endoplasmic reticulum-related and catabolism-related genes were expressed during S. scitamineum infection. A total of 83 genes had homologs in the PHI database, 62 of which correlated with pathogen virulence. A total of 21 proteins had the characteristics of small secreted cysteine-rich proteins (SCRPs), a common source of fungal effectors. The top ten hub genes in each module were identified, and seven were annotated as Mig1-Mig1 protein, glycosyl hydrolase, beta-N-acetylglucosaminidase, secreted chorismate mutase, collagen, mRNA export factor, and pleckstrin homology domain protein, while the remaining three were unknown. Two SCRPs-SPSC_06609 and SPSC_04676-and three proteins-SPSC_01958, SPSC_02155, and SPSC_00940-identified in the PHI database were also among the top ten hub genes in the MEdarkturquoise and MEpurple modules, suggesting that they may play important roles in S. scitamineum infection. A S. scitamineum infection model was postulated based on current findings. These findings help to deepen the current understanding of early events in S. scitamineum infection.

Kinase Hog1 and Adr1 Opposingly Regulate Haploid Cell Morphology by Controlling Vacuole Size in Sporisorium scitamineum.

Morphogenesis is a strictly regulated efficient system in eukaryotes for adapting to environmental changes. However, the morphogenesis regulatory mechanism in smut fungi is not clear. This study reports a relationship between MAP kinase Hog1 and cAMP-dependent protein kinase A catalytic subunit (Adr1) for the morphological regulation in the sugarcane pathogen Sporisorium scitamineum. The results demonstrated that MAP kinase Hog1 and cAMP/PKA signaling pathways are essential for the morphological development of S. scitamineum. Interestingly, MAP kinase Hog1 and cAMP/PKA signaling pathways' defective mutants exhibit an opposite morphological phenotype. The morphology of cAMP/PKA defective mutants is recovered by deleting the SsHOG1 gene. However, MAP kinase Hog1 and cAMP-dependent protein kinase catalytic subunit Adr1 do not interfere with each other. Further investigations showed that kinase Hog1 and Adr1 antagonistically regulates the vacuolar size, which contributes to the cell size and determines the cellular elongation rates. Kinase Hog1 and Adr1 also antagonistically balanced the cell wall integrity and permeability. Taken together, kinase Hog1- and Adr1-based opposing morphogenesis regulation of S. scitamineum by controlling the vacuolar size and cell wall permeability is established during the study.

Aminotransferase SsAro8 Regulates Tryptophan Metabolism Essential for Filamentous Growth of Sugarcane Smut Fungus Sporisorium scitamineum.

Sugarcane smut caused by the basidiomycetous fungus Sporisorium scitamineum leads to severe economic losses globally. Sexual mating/filamentation of S. scitamineum is critical for its pathogenicity, as only the dikaryotic hyphae formed after sexual mating are capable of invading the host cane. Our comparative transcriptome analysis showed that the mitogen-activated protein kinase (MAPK) pathway and the AGC kinase Agc1 (orthologous to yeast Rim15), both governing S. scitamineum mating/filamentation, were induced by elevated tryptophol level, supporting a positive regulation of S. scitamineum mating/filamentation by tryptophol. However, the biosynthesis pathway of tryptophol remains unknown in S. scitamineum. Here, we identified an aminotransferase orthologous to the established tryptophan aminotransferase Tam1/Aro8, catalyzing the first step of tryptophan-dependent indole-3-acetic acid (IAA) production as well as that of the Ehrlich pathway for tryptophol production. We designated this S. scitamineum aminotransferase as SsAro8 and found that it was essential for mating/filamentation. Comparative metabolomics analysis revealed that SsAro8 was involved in tryptophan metabolism, likely for producing important intermediate products, including tryptophol. Exogenous addition of tryptophan or tryptophol could differentially restore mating/filamentation in the ssaro8Δ mutant, indicating that in addition to tryptophol, other product(s) of tryptophan catabolism may also be involved in S. scitamineum mating/filamentation regulation. S. scitamineum could also produce IAA, partially dependent on SsAro8 function. Surprisingly, photodestruction of IAA produced the compound(s) able to suppress S. scitamineum growth/differentiation. Lastly, we found that SsAro8 was required for proper biofilm formation, oxidative stress tolerance, and full pathogenicity in S. scitamineum. Overall, our study establishes the aminotransferase SsAro8 as an essential regulator of S. scitamineum pathogenic differentiation, as well as fungus-host interaction, and therefore of great potential as a molecular target for sugarcane smut disease control. IMPORTANCE Sugarcane smut caused by the basidiomycete fungus S. scitamineum leads to massive economic losses in sugarcane plantation globally. Dikaryotic hyphae formation (filamentous growth) and biofilm formation are two important aspects in S. scitamineum pathogenesis, yet the molecular regulation of these two processes was not as extensively investigated as that in the model pathogenic fungi, e.g., Candida albicans, Ustilago maydis, or Cryptococcus neoformans. In this study, a tryptophan aminotransferase ortholog was identified in S. scitamineum, designated SsAro8. Functional characterization showed that SsAro8 positively regulates both filamentous growth and biofilm formation, respectively, via tryptophol-dependent and -independent manners. Furthermore, SsAro8 is required for full pathogenicity and, thus, is a promising molecular target for designing anti-smut strategy.

Squalene Monooxygenase Gene SsCI80130 Regulates Sporisorium scitamineum Mating/Filamentation and Pathogenicity.

Sugarcane is an important sugar crop and energy crop worldwide. Sugarcane smut caused by Sporisorium scitamineum is a serious fungal disease that occurs worldwide, seriously affecting the yield and quality of sugarcane. It is essential to reveal the molecular pathogenesis of S. scitamineum to explore a new control strategy of sugarcane smut. Based on transcriptome sequencing data of two S. scitamineum strains Ss16 and Ss47, each with a different pathogenicity, our laboratory screened out the SsCI80130 gene predicted to encode squalene monooxygenase. In this study, we obtained the knockout mutants (ΔSs80130+ and ΔSs80130-) and complementary mutants (COM80130+ and COM80130-) of this gene by the polyethylene glycol-mediated (PEG-mediated) protoplast transformation technology, and then performed a functional analysis of the gene. The results showed that the deletion of the SsCI80130 gene resulted in the increased content of squalene (substrate for squalene monooxygenase) and decreased content of ergosterol (the final product of the ergosterol synthesis pathway) in S. scitamineum. Meanwhile, the sporidial growth rate of the knockout mutants was significantly slower than that of the wild type and complementary mutants; under cell-wall stress or oxidative stress, the growth of the knockout mutants was significantly inhibited. In addition, the sexual mating ability and pathogenicity of knockout mutants were significantly weakened, while the sexual mating ability could be restored by adding exogenous small-molecular signal substance cAMP (cyclic adenosine monophosphate) or tryptophol. It is speculated that the SsCI80130 gene was involved in the ergosterol biosynthesis in S. scitamineum and played an important role in the sporidial growth, stress response to different abiotic stresses (including cell wall stress and oxidative stress), sexual mating/filamentation and pathogenicity. Moreover, the SsCI80130 gene may affect the sexual mating and pathogenicity of S. scitamineum by regulating the ergosterol synthesis and the synthesis of the small-molecular signal substance cAMP or tryptophol required for sexual mating. This study reveals for the first time that the gene encoding squalene monooxygenase is involved in regulating the sexual mating and pathogenicity of S. scitamineum, providing a basis for the molecular pathogenic mechanism of S. scitamineum.

Sugarcane Smut: Current Knowledge and the Way Forward for Management.

Whip smut of sugarcane is the most serious and widely spread disease of sugarcane and causes a significant reduction in cane quantity and quality. The severity of this disease often depends on the pathogen races, environmental conditions, cultivar genotype and the interaction among these three factors. Under optimum climatic conditions, this disease has the potential to cause total crop failure. Resistance screening is an ongoing process due to the variability among smut pathogen isolates. Multiple races and mutation ability of smut pathogen makes the breeding task more complex. A number of studies on various aspects of the disease epidemiology and management have been published. Due to many overlapping characteristics within the species complex, there is a dearth of information on early detection and strategies to control the smut pathogen. Furthermore, there is a need to coordinate these findings to expedite its research and control. In this paper, we summarize the disease etiology, especially disease impact on the qualitative and quantitative parameters of sugarcane. We also gathered research progress on molecular-based detection and available information on genetic variability in S.scitamineum. The research on the set of management options needed to effectively cope with the disease are reviewed herein. The present review is expected to be helpful for the further investigation on smut resistance in sugarcane.

SsPEP1, an Effector with Essential Cellular Functions in Sugarcane Smut Fungus.

Biotrophic fungi have to infect their host to obtain nutrients and must establish an interaction with the host to complete their life cycle. In this process, effectors play important roles in manipulating the host's immune system to avoid being attacked. Sporisorium scitamineum is the causative agent of sugarcane smut, the most important disease in sugarcane-producing regions worldwide. In this work, we functionally characterized the conserved effector PEP1 in S. scitamineum. The mating process and the expression of genes in the MAPK signaling pathway and the a and b loci were adversely affected in Sspep1-null mutants. The requirement for SsPEP1 in pathogenicity and symptom development was allele dosage-dependent, i.e., deleting one Sspep1 allele in the mating pair turned a normal black whip with abundant teliospores into a white whip with few teliospores; however, deleting both alleles almost abolished infectivity and whip development. ΔSspep1 mutants produced significantly less mycelium mass within infected plants. Additionally, SsPEP1 was identified as a potent inhibitor of sugarcane POD-1a peroxidase activity, implying that SsPEP1 may function to relieve reactive oxygen species-related stress within the host plant. Taken together, our work demonstrated that SsPEP1 is a multifaceted effector essential for S. scitamineum growth, development, and pathogenicity.