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.

Plant Responses of Maize to Two formae speciales of Sporisorium reilianum Support Recent Fungal Host Jump.

Host jumps are a major factor for the emergence of new fungal pathogens. In the evolution of smut fungi, a putative host jump occurred in Sporisorium reilianum that today exists in two host-adapted formae speciales, the sorghum-pathogenic S. reilianum f. sp. reilianum and maize-pathogenic S. reilianum f. sp. zeae. To understand the molecular host-specific adaptation to maize, we compared the transcriptomes of maize leaves colonized by both formae speciales. We found that both varieties induce many common defense response-associated genes, indicating that both are recognized by the plant as pathogens. S. reilianum f. sp. reilianum additionally induced genes involved in systemic acquired resistance. In contrast, only S. reilianum f. sp. zeae induced expression of chorismate mutases that function in reducing the level of precursors for generation of the defense compound salicylic acid (SA), as well as oxylipin biosynthesis enzymes necessary for generation of the SA antagonist jasmonic acid (JA). In accordance, we found reduced SA levels as well as elevated JA and JA-Ile levels in maize leaves inoculated with the maize-adapted variety. These findings support a model of the emergence of the maize-pathogenic variety from a sorghum-specific ancestor following a recent host jump.

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.

A sugarcane smut fungus effector simulates the host endogenous elicitor peptide to suppress plant immunity.

The smut fungus Sporisorium scitamineum causes the most prevalent disease on sugarcane. The mechanism of its pathogenesis, especially the functions and host targets of its effector proteins, are unknown. In order to identify putative effectors involving in S. scitamineum infection, a weighted gene co-expression network analysis was conducted based on the transcriptome profiles of both smut fungus and sugarcane using a customized microarray. A smut effector gene, termed SsPele1, showed strong co-expression with sugarcane PLANT ELICITOR PEPTIDE RECEPTOR1 (ScPEPR1), which encodes a receptor like kinase for perception of plant elicitor peptide1 (ScPep1). The relationship between SsPele1 and ScPEPR1, and the biological function of SsPele1 were characterized in this study. The SsPele1 C-terminus contains a plant elicitor peptide-like motif, by which SsPele1 interacts strongly with ScPEPR1. Strikingly, the perception of ScPep1 on ScPEPR1 is competed by SsPele1 association, leading to the suppression of ScPEPR1-mediated immune responses. Moreover, the Ustilago maydis effector UmPele1, an ortholog of SsPele1, promotes fungal virulence using the same strategy. This study reveals a novel strategy by which a fungal effector can mimic the plant elicitor peptide to complete its perception and attenuate receptor-activated immunity.

Increase in cytotoxic lignans production after smut infection in sugar cane plants.

Smut infection alters the transcription of dirigent proteins (DIR) by sugarcane plants. Here, we show that these alterations are associated to an elevated production of cytotoxic lignans. Smut-resistant sugarcane varieties display a fivefold increase in pinoresinol and also produce elevated amounts of secoisolariciresinol. Conversely, smut-sensitive varieties do not produce pinoresinol or secoisolariciresinol upon infection, synthesizing instead small amounts of matairesinol. Our data indicate that commercial pinoresinol and secoisolariciresinol seem to prevent smut teliospore germination and sporidia release from sprouted teliospores. Consistently, we observed abundant morphological alterations of sporidia incubated in the presence of these lignans. However, commercial lignans do not block the development of the pathogen in a definitive way. Additional experiments demonstrate that only the extracts from healthy or smut-exposed resistant plants inhibit sporidia growth in vitro, indicating that a specific mixture of lignans from resistant plants is necessary to constitute an effective defense mechanism.

Polyamines levels increase in smut teliospores after contact with sugarcane glycoproteins as a plant defensive mechanism.

Previous studies have already highlighted the correlation between Sporisorium scitamineum pathogenicity and sugarcane polyamine accumulation. It was shown that high infectivity correlates with an increase in the amount of spermidine, spermine and cadaverine conjugated to phenols in the sensitive cultivars whereas resistant plants mainly produce free putrescine. However, these previous studies did not clarify the role of these polyamides in the disorders caused to the plant. Therefore, the purpose of this research is to clarify the effect of polyamines on the development of smut disease. In this paper, commercial polyamines were firstly assayed on smut teliospores germination. Secondly, effects were correlated to changes in endogenous polyamines after contact with defense sugarcane glycoproteins. Low concentrations of spermidine significantly activated teliospore germination, while putrescine had no activating effect on germination. Interestingly, it was observed that the diamine caused nuclear decondensation and breakage of the teliospore cell wall whereas the treatment of teliospores with spermidine did not induce nuclear decondensation or cell wall breakdown. Moreover, the number of polymerized microtubules increased in the presence of 7.5 mM spermidine but it decreased with putrescine which indicates that polyamines effects on Sporisorium scitamineum teliospore germination could be mediated through microtubules interaction. An increased production of polyamines in smut teliospores has been related to sugarcane resistance to the disease. Teliospores incubation with high molecular mass glycoproteins (HMMG) from the uninoculated resistant variety of sugarcane, Mayari 55-14, caused an increase of the insoluble fraction of putrescine, spermidine and spermine inside the teliospore cells. Moreover, the level of the soluble fraction of spermidine (S fraction) increased inside teliospores and the excess was released to the medium. The HMMG glycoproteins purified from Mayarí 55-14 plants previously inoculated with the pathogen significantly increased the levels of both retained and secreted soluble putrescine and spermidine. Polyamines levels did not increase in teliospores after incubation with HMMG produced by non resistant variety Barbados 42231 which could be related to the incapacity of these plants to defend themselves against smut disease. Thus, a hypothesis about the role of polyamines in sugarcane-smut interaction is explained.

A dynamic degradome landscape on miRNAs and their predicted targets in sugarcane caused by Sporisorium scitamineum stress.

BACKGROUND: Sugarcane smut is a fungal disease caused by Sporisorium scitamineum. Cultivation of smut-resistant sugarcane varieties is the most effective way to control this disease. The interaction between sugarcane and S. scitamineum is a complex network system. However, to date, there is no report on the identification of microRNA (miRNA) target genes of sugarcane in response to smut pathogen infection by degradome technology. RESULTS: TaqMan qRT-PCR detection and enzyme activity determination showed that S. scitamineum rapidly proliferated and incurred significant enzyme activity changes in the reactive oxygen species metabolic pathway and phenylpropanoid metabolic pathway at 2 d and 5 d after inoculation, which was the best time points to study target gene degradation during sugarcane and S. scitamineum interaction. A total of 122.33 Mb of raw data was obtained from degradome sequencing analysis of YC05-179 (smut-resistant) and ROC22 (smut-susceptible) after inoculation. The Q30 of each sample was > 93%, and the sequence used for degradation site analysis exactly matched the sugarcane reference sequence. A total of 309 target genes were predicted in sugarcane, corresponding to 97 known miRNAs and 112 novel miRNAs, and 337 degradation sites, suggesting that miRNAs can efficiently direct cleavage at multiple sites in the predicted target mRNAs. Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that the predicted target genes were involved in various regulatory processes, such as signal transduction mechanisms, inorganic ion transport and metabolism, defense mechanisms, translation, posttranslational modifications, energy production and conversion, and glycerolipid metabolism. qRT-PCR analysis of the expression level of 13 predicted target genes and their corresponding miRNAs revealed that there was no obvious negative regulatory relationship between miRNAs and their target genes. In addition, a number of putative resistance-related target genes regulated by miRNA-mediated cleavage were accumulated in sugarcane during S. scitamineum infection, suggesting that feedback regulation of miRNAs may be involved in the response of sugarcane to S. scitamineum infection. CONCLUSIONS: This study elucidates the underlying response of sugarcane to S. scitamineum infection, and also provides a resource for miRNAs and their predicted target genes for smut resistance improvement in sugarcane.

Neofunctionalization of the secreted Tin2 effector in the fungal pathogen Ustilago maydis.

Plant-pathogenic fungi hijack their hosts by secreting effector proteins. Effectors serve to suppress plant immune responses and modulate the host metabolism to benefit the pathogen. Smut fungi are biotrophic pathogens that also parasitize important cereals, including maize1. Symptom development is usually restricted to the plant inflorescences. Ustilago maydis is an exception in its ability to cause tumours in both inflorescences and leaves of maize, and in inducing anthocyanin biosynthesis through the secreted Tin2 effector2,3. How the unique lifestyle of U. maydis has evolved remains to be elucidated. Here we show that Tin2 in U. maydis has been neofunctionalized. We functionally compared Tin2 effectors of U. maydis and the related smut Sporisorium reilianum, which results in symptoms only in the inflorescences of maize and fails to induce anthocyanin. We show that Tin2 effectors from both fungi target distinct paralogues of a maize protein kinase, leading to stabilization and inhibition, respectively. An ancestral Tin2 effector functionally replaced the virulence function of S. reilianum Tin2 but failed to induce anthocyanin, and was unable to substitute for Tin2 in U. maydis. This shows that Tin2 in U. maydis has acquired a specialized function, probably connected to the distinct pathogenic lifestyle of this fungus.

Antagonistic yeasts from a salt-lake region in Egypt: identification of a taxonomically distinct group of phylloplane strains related to Sporisorium.

Non-pathogenic yeasts antagonising microorganisms that cause pre- and postharvest diseases of plants have been found in diverse habitats. Their practical applicability as biocontrol agents (BCAs) depends on the strength of their antagonistic activity and/or spectrum of sensitive target microorganisms. In this study, yeasts were isolated from the phylloplane and fruits of plants growing in the alkaline water lake region Wadi El-Natrun, Egypt, and tested for antifungal and antibacterial activity. All phylloplane yeast isolates belonged to the Basidiomycota and most of them could antagonise at least certain test organisms. One group of isolates showing strong antagonism against almost all fungi and yeasts appears to represent a hitherto undescribed species distantly related to the smut genus Sporisorium. This is the first report of antagonistic activity in Sporisorium. The isolates assigned to Naganishia and Papiliotrema were more effective against bacteria. The broadest range and intensity of antagonism was observed in the fruit-associated strains belonging to the ascomycetous species Wickerhamomyces subpelliculosus. The Wickerhamomyces strains are good broad-spectrum BCA candidates, the Sporisorium strains could be used as efficient antifungal BCAs, whereas the Papiliotrema isolate can be exploited as an antibacterial biocontrol agent.

cAMP/PKA signalling pathway regulates redox homeostasis essential for Sporisorium scitamineum mating/filamentation and virulence.

The fungal pathogen Sporisorium scitamineum causes sugarcane smut disease. The formation and growth of dikaryotic hypha after sexual mating is critical for S. scitamineum pathogenicity, however regulation of S. scitimineum mating has not been studied in detail. We identified and characterized the core components of the conserved cAMP/PKA pathway in S. scitamineum by reverse genetics. Our results showed that cAMP/PKA signalling pathway is essential for proper mating and filamentation, and thus critical for S. scitamineum virulence. We further demonstrated that an elevated intracellular ROS (reactive oxygen species) level promotes S. scitamineum mating-filamentation, via transcriptional regulation of ROS catabolic enzymes, and is under regulation of the cAMP/PKA signalling pathway. Furthermore, we found that fungal cAMP/PKA signalling pathway is also involved in regulation of host ROS response. Overall, our work displayed a positive role of elevated intracellular ROS in fungal differentiation and virulence.

Disease severity enhancement by an esterase from non-phytopathogenic yeast Pseudozyma antarctica and its potential as adjuvant for biocontrol agents.

The phylloplane yeast Pseudozyma antarctica secretes an esterase, named PaE, and xylanase when cultivated with xylose. We previously observed that the lipophilic layer of Micro-Tom tomato leaves became thinner after the culture filtrate treatment. The leaves developed reduced water-holding ability and became wilted. In this study, the purified enzymes were spotted on Micro-Tom leaves. PaE, but not xylanase, thinned the lipophilic layer of leaves and decreased leaf resistance to the phytopathogenic fungus Botrytis cinerea. Disease severity increased significantly in detached leaves and potted plants treated with the culture filtrate and B. cinerea spores compared with those treated with inactivated enzyme and B. cinerea alone. Spore germination ratios, numbers of penetrating fungal hyphae in the leaves, and fungal DNA contents also increased significantly on the detached leaves. Japanese knotweed (Fallopia japonica), a serious invasive alien weed in Europe and North America, also became susceptible to infection by the rust pathogen Puccinia polygoni-amphibii var. tovariae following the culture filtrate treatment. The culture filtrate treatment increased disease development in plants induced by both phytopathogenic fungi. Our results suggest that P. antarctica culture filtrate could be used as an adjuvant for sustainable biological weed control using phytopathogenic fungi.

The Role of Sugarcane Catalase Gene ScCAT2 in the Defense Response to Pathogen Challenge and Adversity Stress.

Catalases, which consist of multiple structural isoforms, catalyze the decomposition of hydrogen peroxide in cells to prevent membrane lipid peroxidation. In this study, a group II catalase gene ScCAT2 (GenBank Accession No. KF528830) was isolated from sugarcane genotype Yacheng05-179. ScCAT2 encoded a predicted protein of 493 amino acid residues, including a catalase active site signature (FARERIPERVVHARGAS) and a heme-ligand signature (RVFAYADTQ). Subcellular localization experiments showed that the ScCAT2 protein was distributed in the cytoplasm, plasma membrane, and nucleus of Nicotiana benthamiana epidermal cells. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that the ScCAT2 gene was ubiquitously expressed in sugarcane tissues, with expression levels from high to low in stem skin, stem pith, roots, buds, and leaves. ScCAT2 mRNA expression was upregulated after treatment with abscisic acid (ABA), sodium chloride (NaCl), polyethylene glycol (PEG), and 4 °C low temperature, but downregulated by salicylic acid (SA), methyl jasmonate (MeJA), and copper chloride (CuCl2). Moreover, tolerance of Escherichia coli Rosetta cells carrying pET-32a-ScCAT2 was enhanced by NaCl stress, but not by CuCl2 stress. Sporisorium scitamineum infection of 10 different sugarcane genotypes showed that except for YZ03-258, FN40, and FN39, ScCAT2 transcript abundance in four smut-resistant cultivars (Yacheng05-179, YZ01-1413, YT96-86, and LC05-136) significantly increased at the early stage (1 day post-inoculation), and was decreased or did not change in the two smut-medium-susceptibility cultivars (ROC22 and GT02-467), and one smut-susceptible cultivar (YZ03-103) from 0 to 3 dpi. Meanwhile, the N. benthamiana leaves that transiently overexpressed ScCAT2 exhibited less severe disease symptoms, more intense 3,3'-diaminobenzidine (DAB) staining, and higher expression levels of tobacco immune-related marker genes than the control after inoculation with tobacco pathogen Ralstonia solanacearum or Fusarium solani var. coeruleum. These results indicate that ScCAT2 plays a positive role in immune responses during plant⁻pathogen interactions, as well as in salt, drought, and cold stresses.

Transcriptional analysis identifies major pathways as response components to Sporisorium scitamineum stress in sugarcane.

BACKGROUND: Sugarcane smut, which is caused by Sporisorium scitamineum, is a severe fungal disease affecting sugarcane. However, the major pathways involved in the interaction between sugarcane and S. scitamineum remains unclear. RESULTS: In the present study, suppression subtractive hybridization (SSH) library construction, together with reverse northern blotting, was conducted on the most prevalent sugarcane genotype ROC22 challenged with S. scitamineum. After alignment and homologous expressed sequence tag (EST) assembly, a total of 155 differentially expressed unigenes were identified from SSH libraries. Totally, 26 of 155 differentially expressed unigenes were analyzed by qRT-PCR in sugarcane smut-resistant genotype YC05-179 and susceptible genotype ROC22. Genes encoded two unknown protein (Q1 and Q11), serine/threonine kinase (Q2), fiber protein (Q3), eukaryotic translation initiation factor 5A (Q23), and Sc14-3-3-like protein (Q24) were induced in sugarcane smut-resistant genotype YC05-179 but inhibited in susceptible genotype ROC22. Based on the differential expression data achieved from SSH libraries and qRT-PCR, we found that, serine/threonine kinases, Ca2+ sensors, mitogen-activated protein genes and some NBS-LRR genes may involve in the signal recognition and transduction of smut fungus infection in sugarcane. While in the plant hormone signaling pathways, the genes related to auxin, abscisic acid, salicylic acid and ethylene were more apparently in response to smut fungus invasion. The hypersensitive response, protein metabolism, polyamine synthesis, and cell wall formation may play an important role in sugarcane defense against smut fungus colonization. Additionally, the Sc14-3-3 might serve as a molecular modulator in sugarcane being immune to smut disease by interacting with proteins like ScGAPN (Q10), which have been further verified by BiFC assay. CONCLUSIONS: The findings of the present study could provide a general view about gene pathways involving in sugarcane defense against smut disease and facilitate a better understanding of the molecular mechanism underlying sugarcane-S. scitamineum interaction.

Morphological differences between aerial and submerged sporidia of bio-fongicide Pseudozyma flocculosa CBS 16788.

Pseudozyma flocculosa is a fungus very useful and highly efficient as a biocontrol agent against powdery mildew. The reproduction of this fungus occurs exclusively by asexual production of conidia or sporidia that are the most suitable form for agricultural use and seems to be the most resistant to storage conditions. Despite the advantages offered by P. flocculosa in biological control, the use of this fungus use remains largely limited compared to that of chemical fungicides, at least partly due to the difficulty to obtain sporidia resistant to adverse environmental stresses in submerged culture conditions. Under solid-state and submerged-state cultivation, P. flocculosa strain CBS 16788 produced different types of sporidia. The submerged sporidia (SS) appeared relatively uniform in size, which was 15,4 ± 1,6 µm µm long, and 2,8 ± 0.8 µm wide. The aerial sporidia (AS) varied in shape and size, with a mean length of 8,2 ± 3 µm and width of 2,3 ± 0.6 µm. Under scanning and transmission electron microscopy, the cell wall of submerged sporidia was thinner than that of aerial spores, and the surface was smooth in contrast to the aerial sporidia that had a tendency to have verrucous, brittle surface characteristics. The thickness of the aerial sporidia wall is due to the presence of an outer layer rich in melanin. The sporidia germination was compared on YMPD (yeast extract, malt extract, soy peptone, dextrose and agar) coated coverslips. The aerial sporidia did not show germ tubes until 5 h of incubation, while the submerged sporidia showed many germ tubes after the same time. The resistance against the adverse environmental conditions in relation to the type of sporidia of P. flocculosa is discussed.

Differential expression of SofDIR16 and SofCAD genes in smut resistant and susceptible sugarcane cultivars in response to Sporisorium scitamineum.

Proteomic profiling of the stalk of a smut resistant and a susceptible sugarcane cultivars revealed the presence of dirigent and dirigent-like proteins in abundance in the pool of high molecular mass (HMMG) and mid-molecular mass (MMMG) glycoproteins, produced as part of the defensive response to the fungal smut pathogen. Quantitative RT-PCR analysis showed that expression levels of SofDIR16 (sugarcane dirigent16) and SofCAD (sugarcane cinnamyl alcohol dehydrogenase) were higher in the smut resistant My 55-14 cultivar than in the sensitive B 42231 cultivar prior to infection. Inoculation with fungal sporidia or water decreased the level of SofCAD transcripts in My 55-14, indicating that regulation of SofCAD expression does not take part of the specific response to smut infection. In contrast, SofDIR16 expression was almost nullified in My 55-14 after inoculation with fungal sporidia, but not after water injection. It is proposed that the decreased expression of dirigent proteins induces the formation of lignans, which are involved in the defense response of the smut resistant My 55-14 cultivar.

A sugarcane pathogenesis-related protein, ScPR10, plays a positive role in defense responses under Sporisorium scitamineum, SrMV, SA, and MeJA stresses.

KEY MESSAGE: A pathogenesis-related gene, ScPR10 , was isolated from sugarcane and its bio-function was characterized, demonstrating that ScPR10 was involved in plant defense responses to Sporisorium scitamineum , SrMV, SA, and MeJA stresses. Plant fungal and viral diseases are the major concerns in sugarcane industry. Many anti-fungal and antivirus components, including pathogenesis-related (PR) proteins, have been identified. The pathogenesis-related protein 10 (PR10) is the dominant group in PR families, involved in the plant defense mechanism. In this study, ScPR10 (GenBank Acc. No. KT887884), a 701-bp-length PR10 gene with a 483 bp-length open reading frame, was isolated from sugarcane. Its transient expression in the leaves of Nicotiana benthamiana indicated that the function role of ScPR10 is likely in the nucleus, and it increased the level of H2O2 accumulation in leaf cells. Moreover, ScPR10 could also enhance the resistance of N. benthamiana leaves to infection by Pseudomonas solanacearum and Fusarium solani var. coeruleum. Quantitative real-time PCR analysis revealed that ScPR10 was not constitutively expressed in sugarcane tissues due to its high expression in the buds and scant presence in root tips. In addition, the transcript of ScPR10 could be induced by a pathogenic fungus (Sporisorium scitamineum) and a virus (Sorghum mosaic virus, SrMV) in the resistant sugarcane cultivars, while it was down-regulated in the susceptible ones. After exposure to salicylic acid (SA) and methyl jasmonate (MeJA), ScPR10 peaked at 6 and 12 h, respectively. These results suggest that ScPR10 can play a positive role in sugarcane defense responses to S. scitamineum, SrMV, SA, and MeJA stresses.

Small RNA sequencing reveals a role for sugarcane miRNAs and their targets in response to Sporisorium scitamineum infection.

BACKGROUND: Sugarcane smut caused by Sporisorium scitamineum leads to a significant reduction in cane yield and sucrose content. MicroRNAs (miRNAs) play an important role in regulating plant responses to biotic stress. The present study was the first to use two sugarcane genotypes, YA05-179 (smut-resistant) and ROC22 (smut-susceptible), to identify differentially expressed miRNAs in sugarcane challenged with S. scitamineum by using high-throughput sequencing. RESULTS: The predicted target gene number corresponding to known differentially expressed miRNAs in YA05-179 was less than that in ROC22, however most of them were in common. Expression of differential miRNAs under S. scitamineum challenge was mostly downregulated, with similar trends in the two varieties. Gene ontology (GO) analysis showed that the target gene classification of known miRNAs was similar to that of the newly identified miRNAs. These were mainly associated with cellular processes and metabolic processes in the biological process category, as well as combination and catalytic activity in the molecular function category. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that these predicted target genes involved in a series of physiological and biochemical pathways or disease resistance-related physiological metabolism and signal transduction pathways, suggesting that the molecular interaction mechanism between sugarcane and S. scitamineum was a complex network system. These findings also showed certain predicted target genes of miR5671, miR5054, miR5783, miR5221, and miR6478 play roles in the mitogen-activated protein kinase (MAPK) signaling pathway, plant hormone signal transduction, and plant-pathogen interaction. Quantitative real-time PCR (qRT-PCR) analysis showed that majority of the known miRNAs and its predicted target genes followed a negatively regulated mode. Seven out of eight predicted target genes showed identical expression after 12 h treatment and reached the highest degree of matching at 48 h, indicating that the regulatory role of miRNAs on the target genes in sugarcane was maximized at 48 h after S. scitamineum challenge. CONCLUSIONS: Taken together, our findings serve as evidence for the association of miRNA expression with the molecular mechanism underlying the pathogenesis of sugarcane smut, particularly on the significance of miRNA levels in relation to the cultivation of smut-resistant sugarcane varieties.

Cytological and Molecular Characterization of ZmWAK-Mediated Head-Smut Resistance in Maize.

Head smut, caused by the fungal pathogen Sporisorium reilianum, poses a threat to maize production worldwide. ZmWAK, a cell wall-associated receptor kinase, confers quantitative resistance to head smut disease. Here, two near-isogenic lines (NILs), susceptible line Huangzao4 and its ZmWAK-converted resistant line Huangzao4R, were used to decipher the role of ZmWAK in head smut resistance. Cytological and molecular characterization in response to S. reilianum infection was compared between two NILs. Upon S. reilianum infection, the growth of pathogen hyphae was severely arrested in the ZmWAK-converted resistant line Huangzao4R, relative to its susceptible parental line Huangzao4. Infected cells exhibited apoptosis-like features in Huangzao4R and hyphae were sequestered within dead cells, whereas pathogen invasion caused autophagy in Huangzao4, which failed to prevent hyphal spreading. Integrated transcriptomic and metabolomic analysis indicated that ZmWAK functions as a hub in the trade-off between growth and defense, whereby ZmWAK promotes cell growth in the absence of the pathogen and switches to a defense response upon S. reilianum attack. These findings shed light on an elegant regulatory mechanism governed by ZmWAK in the trade-off between growth and head smut defense.

Sugarcane smut: shedding light on the development of the whip-shaped sorus.

Background and Aims: Sugarcane smut is caused by the fungus Sporisorium scitamineum (Ustilaginales/Ustilaginomycotina/Basidiomycota), which is responsible for losses in sugarcane production worldwide. Infected plants show a profound metabolic modification resulting in the development of a whip-shaped structure (sorus) composed of a mixture of plant tissues and fungal hyphae. Within this structure, ustilospores develop and disseminate the disease. Despite the importance of this disease, a detailed histopathological analysis of the plant-pathogen interaction is lacking. Methods: The whip-shaped sorus was investigated using light microscopy, scanning and transmission electron microscopy, histochemical tests and epifluorescence microscopy coupled with deconvolution. Key Results: Sorus growth is mediated by intercalary meristem activity at the base of the sorus, where the fungus causes partial host cell wall degradation and formation of intercellular spaces. Sporogenesis in S. scitamineum is thallic, with ustilospore initials in intercalary or terminal positions, and mostly restricted to the base of the sorus. Ustilospore maturation is centrifugal in relation to the ground parenchyma and occurs throughout the sorus median region. At the apex of the sorus, the fungus produces sterile cells and promotes host cell detachment. Hyphae are present throughout the central axis of the sorus (columella). The plant cell produces callose around the intracellular hyphae as well as inside the papillae at the infection site. Conclusions: The ontogeny of the whip-shaped sorus suggests that the fungus can cause the acropetal growth in the intercalary meristem. The sporogenesis of S. scitamineum was described in detail, demonstrating that the spores are formed exclusively at the base of the whip. Light was also shed on the nature of the sterile cells. The presence of the fungus alters the host cell wall composition, promotes its degradation and causes the release of some peripheral cells of the sorus. Finally, callose was observed around fungal hyphae in infected cells, suggesting that deposition of callose by the host may act as a structural response to fungal infection.

Defence sugarcane glycoproteins disorganize microtubules and prevent nuclear polarization and germination of Sporisorium scitamineum teliospores.

Microtubules (MTs) are involved in the germination of Sporisorium scitamineum teliospores. Resistant varieties of sugar cane plants produce defence glycoproteins that prevent the infection of the plants by the filamentous fungi Sporisorium scitamineum. Here, we show that a fraction of these glycoproteins prevents the correct arrangement of MTs and causes nuclear fragmentation defects. As a result, nuclei cannot correctly migrate through the growing hyphae, causing germinative failure. Arginase activity contained in defence glycoproteins is already described for preventing fungal germination. Now, its enzymatically active form is presented as a link between the defensive capacity of glycoproteins and the MT disorganization in fungal cells. Active arginase is produced in healthy and resistant plants; conversely, it is not detected in the juice from susceptible varieties, which explains why MT depolarization, nuclear disorganization as well as germination of teliospores are not significantly affected by glycoproteins from non-resistant plants. Our results also suggest that susceptible plants try to increase their levels of arginase after detecting the presence of the pathogen. However, this signal comes "too late" and such defensive mechanism fails.