False Smut Balls Research Articles

The bZIP Transcription Factor UvbZIP14 Regulates Vegetative Growth, Conidiation and Abiotic Stress Tolerance in Ustilaginoidea virens

ABSTRACTbZIP transcription factors play a crucial role in regulating various physiological processes in phytopathogens. Ustilaginoidea virens is known to possess 28 bZIP transcription factors; however, their biological functions remain unexplored. In this study, the function of UvbZIP14 in U. virens was investigated. The expression levels of UvbZIP14 were found to be up‐regulated after rice inoculation with U. virens. The knockout mutants and complemented strains of UvbZIP14 were generated and characterised. The knockout mutants display increased growth and decreased conidiation. Compared with the wild‐type strain, the knockout mutants exhibit reduced sensitivity to NaCl and sorbitol but increased sensitivity to CFW. There was no significant difference in the number of false smut balls in rice between the knockout and wild‐type strains. These results findings highlight the significant role of UvbZIP14 in regulating vegetative growth, conidiation and tolerance to abiotic stress in U. virens.

UvHOS3-mediated histone deacetylation is essential for virulence and negatively regulates ustilaginoidin biosynthesis in Ustilaginoidea virens.

Ustilaginoidea virens is the causal agent of rice false smut, which has recently become one of the most important rice diseases worldwide. Ustilaginoidins, a major type of mycotoxins produced in false smut balls, greatly deteriorates grain quality. Histone acetylation and deacetylation are involved in regulating secondary metabolism in fungi. However, little is yet known on the functions of histone deacetylases (HDACs) in virulence and mycotoxin biosynthesis in U. virens. Here, we characterized the functions of the HDAC UvHOS3 in U. virens. The ΔUvhos3 deletion mutant exhibited the phenotypes of retarded growth, increased mycelial branches and reduced conidiation and virulence. The ΔUvhos3 mutants were more sensitive to sorbitol, sodium dodecyl sulphate and oxidative stress/H2 O2 . ΔUvhos3 generated significantly more ustilaginoidins. RNA-Seq and metabolomics analyses also revealed that UvHOS3 is a key negative player in regulating secondary metabolism, especially mycotoxin biosynthesis. Notably, UvHOS3 mediates deacetylation of H3 and H4 at H3K9, H3K18, H3K27 and H4K8 residues. Chromatin immunoprecipitation assays indicated that UvHOS3 regulates mycotoxin biosynthesis, particularly for ustilaginoidin and sorbicillinoid production, by modulating the acetylation level of H3K18. Collectively, this study deepens the understanding of molecular mechanisms of the HDAC UvHOS3 in regulating virulence and mycotoxin biosynthesis in phytopathogenic fungi.

Water Extract of Rice False Smut Balls Activates Nrf2/HO-1 and Apoptosis Pathways, Causing Liver Injury

Ustiloxins are vital cyclopeptide mycotoxins originally isolated from rice false smut balls that form in rice spikelets infected by the fungal pathogen Ustilaginoidea virens. The toxicity of the water extract of rice false smut balls (RBWE) remains to be investigated. Studies have shown that RBWE may be toxic to animals, but toxicological evidence is still lacking. In this study, we found that the IC50 values of RBWE to BNL CL.2 cells at 24 and 48 h were 40.02 and 30.11 μg/mL, respectively, with positive correlations with dose toxicity and time toxicity. After treatment with RBWE, the number of BNL CL.2 cells decreased significantly, and the morphology of BNL CL.2 cells showed atrophy and wall detachment. RBWE induced DNA presynthesis phase arrest of BNL CL.2 cells, increased the proportion of apoptotic cells and inhibited cell proliferation. RBWE up-regulated reactive oxygen species (ROS) levels and lowered mitochondrial membrane potentials. Additionally, Western blot and qRT-PCR results suggested that RBWE exerted the above effects by promoting the Nrf2/HO-1 and caspase-induced apoptosis pathways in vitro and in vivo. The contents of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and total bile acids in the serum of mice from Institute of Cancer were significantly up-regulated by RBWE. At the same time, RBWE can lead to increases in ROS and malondialdehyde contents, decreases in contents of oxidized glutathione, glutathione and reduced glutathione, as well as decrease in catalase and superoxide dismutase activities in mouse liver tissues, demonstrating that oxidative stress occurred in mice. Moreover, liver damage was further detected by haematoxylin-eosin staining and electron microscopy to verify the damage to the mice caused by RBWE. In general, RBWE may cause hepatotoxicity in vivo and in vitro via the apoptosis pathway, which provides a reference for hepatotoxicity and its mechanism of action.

Visualization of the entire process of rice spikelet infection by Ustilaginoidea virens through nondestructive inoculation.

Rice false smut caused by Ustilaginoidea virens, is a destructive fungal disease encountered in many rice-producing areas worldwide. To determine the process by which U. virens infects rice spikelets in the field. The green fluorescent protein-labeled U. virens was used as an inoculum to conduct artificial inoculation on rice at the booting stage via non-destructive panicle sheath instillation inoculation. The results showed that the conidia of U. virens germinated on the surface of rice glumes and produced hyphae, which clustered at the mouth of rice glumes and entered the glumes through the gap between the palea and lemma. The conidia of U. virens colonized in rice floral organs, which led to pollen abortion of rice. U. virens wrapped the whole rice floral organ, and the floral organ-hyphae complex gradually expanded to open the glumes to form a rice false smut ball, which was two to three times larger than that observed in normal rice. Panicle sheath instillation inoculation was shown to be a non-destructive inoculation method that could simulate the natural infection of U. virens in the field. The entire infection process of U. virens was visualized, providing a theoretical reference for formulating strategies to control rice false smut in the field.

Improving disease resistance to rice false smut without yield penalty by manipulating the expression of effector target

Rice grain production and quality are threatened by many devastating diseases. False smut caused by Ustilaginoidea virens is emerging as a major fungal disease of rice, leading to a severe loss of yield. U. virens invades only rice panicles at the late stage of booting and replaces grains with false smut balls containing mycotoxins, which endanger human and animal health (Sun et al., 2020). This successful colonization is accomplished through manipulating host immune responses by U. virens effectors.

The Adaptor Protein UvSte50 Governs Fungal Pathogenicity of Ustilaginoidea virens via the MAPK Signaling Pathway.

The mitogen-activated protein kinase (MAPK) signaling pathways regulate diverse cellular processes and have been partially characterized in the rice false smut fungus Ustilaginoidea virens. UvSte50 has been identified as a homolog to Saccharomyces cerevisiae Ste50, which is known to be an adaptor protein for MAPK cascades. ΔUvste50 was found to be defective in conidiation, sensitive to hyperosmotic and oxidative stresses, and non-pathogenic. The mycelial expansion of ΔUvste50 inside spikelets of rice terminated at stamen filaments, eventually resulting in a lack of formation of false smut balls on spikelets. We determined that UvSte50 directly interacts with both UvSte7 (MAPK kinase; MEK) and UvSte11 (MAPK kinase kinase; MEKK), where the Ras-association (RA) domain of UvSte50 is indispensable for its interaction with UvSte7. UvSte50 also interacts with UvHog1, a MAP kinase of the Hog1-MAPK pathway, which is known to have important roles in hyphal growth and stress responses in U. virens. In addition, affinity capture–mass spectrometry analysis and yeast two-hybrid assay were conducted, through which we identified the interactions of UvSte50 with UvRas2, UvAc1 (adenylate cyclase), and UvCap1 (cyclase-associated protein), key components of the Ras/cAMP signaling pathway in U. virens. Together, UvSte50 functions as an adaptor protein interacting with multiple components of the MAPK and Ras/cAMP signaling pathways, thus playing critical role in plant infection by U. virens.

The bZIP transcription factor UvbZIP6 mediates fungal growth, stress response, and false smut formation in Ustilaginoidea virens

Rice false smut, caused by Ustilaginoidea virens, is one of the most destructive diseases in major world rice-producing regions. Basic leucine zipper (bZIP) proteins, which belong to an evolutionarily conserved transcription factor family and play critical roles in various biological processes in eukaryotes, have been previously identified in U. virens; however, their functions still need to be further elucidated. Therefore, we aimed to analyze the biological roles of UvbZIP6, a member of the bZIP family in U. virens. In this study, we found that UvbZIP6 was highly up-regulated at 7 days post-inoculation. Deletion of UvbZIP6 in U. virens resulted in an increase in fungal growth and sensitivity to Congo red and calcofluor white, whereas a decrease in sensitivity to hyperosmotic, oxidative, and sodium dodecyl sulfate stresses. Conidiation capacity was reduced in UvbZIP6-knockout mutants, but conidial morphology and germination were not affected. Although UvbZIP6-knockout mutants caused infection in rice plants, they could not form false smut balls. Our study indicates that UvbZIP6 is required for fungal growth, conidiation, stress response, and false smut ball formation of U. virens.

The Velvet Protein UvVEA Regulates Conidiation and Chlamydospore Formation in Ustilaginoidea virens.

Rice false smut, caused by Ustilaginoidea virens, is a serious disease of rice worldwide, severely reducing the quantity and quality of rice production. The conserved fungal velvet proteins are global regulators of diverse cellular processes. We identified and functionally characterized two velvet genes, UvVEA and UvVELB, in U. virens. The deletion of these genes affected the conidiation of U. virens but had no effect on the virulence of this pathogen. Interestingly, the ΔUvVEA mutants appeared in the form of smaller false smut balls with a reduced number of chlamydospores compared with the wide-type strains. In addition, the deletion of UvVEA affected the expression of some transmembrane transport genes during chlamydospore formation and rice false smut balls development. Furthermore, the ΔUvVEA mutants were shown to be defective in the utilization of glucose. These findings proved the regulatory mechanism underlying the formation of rice false smut balls and chlamydospores and provided a basis for the further exploration of the mechanism of these processes.

Identification of potential donors for false smut resistance in elite breeding lines of rice (Oryza sativaL.) under field conditions

AbstractFalse smut of rice is an emerging disease and caused severe damage to hybrids and inbred rice cultivars grown in Asian countries. The objective of the study was to quantify of false smut resistance and identification of donors in some of the advanced breeding lines and rice varieties developed at Punjab Agricultural University, Ludhiana, India. A total of 31 genotypes were evaluated for three years in two planting date per year under field conditions. The lines were categorized into short, medium and long durations based on days to flowering. False smut was quantified using different disease variables such as per cent infected panicle, number of false smut ball per plant and disease score. Disease variables were significantly and positively correlated to each other. The infected panicle ranged 0.0–75.4% was observed among the genotypes. Three advanced lines namely RGS-2 (short), RGM-3 (medium) and RGL-3 (long) showed the lowest ranged 0.0–4.9% of infected panicle as compared to susceptible checks (47.7–75.4%). The genotypes were divided into five groups according to a component of resistance. The third group had the lowest average values (3.3%) of infected panicle as compared to the fifth group with the highest values (36.2%) of the infected panicle. The overall trend of disease variables was higher in short duration genotypes as compared to medium and long durations. Weather factors such as rain fall, rainy days and high relative humidity during the flowering period were favourable for disease development. The genotypes with lower disease variables could be utilized in diseases resistance breeding programme.

Autophagy-related protein UvAtg7 contributes to mycelial growth, virulence, asexual reproduction and cell stress response in rice false smut fungus Ustilaginoidea virens

Autophagy is a conserved mechanism for nutrient and cytoplasmic components recycling in eukaryotic cell, in which E1-like enzyme Atg7 activates ubiquitin-like conjugation in the autophagy pathway. In plant pathogenic fungi Ustilaginoidea virens, UvAtg7, an ortholog of AAtg7 in baker’s yeast was identified and functionally investigated. UvAtg7 was confirmed to be essential for autophagy, because the disruption of UvATG7 gene in U. virens completely blocked the fusion of autophagosome-like into vacuoles and catalytic degradation of GFP-UvAtg8 under N-starving condition. The fluorescent signal indicated UvAtg7 protein was dispersed in cytoplasma, but spatially coordinated with core autophagy protein UvAtg8 on occasion. Interestingly, disruption of UvATG7 in U. virens caused slightly reduction in mycelial growth, but resulted in a considerable decrease in virulence, conidia production in YT broth and chlamydospore formation on rice false smut balls. Moreover, the UvATG7 deletion mutants exhibited increased sensitivity to cell wall integrity stress caused by congo red and calcofluor white, meanwhile the UvATG7 deletion mutants showed decreased sensitivity to osmotic stress, cell membrane stress and reactiveoxygen stress caused by sorbitol, sodium dodecyl sulfate and H2O2, respectively. All of these defects in UvATG7 deletion mutants could be partially or completely restored by gene complementation. In general, our study indicates that UvAtg7 is essential in autophagy pathway and contributes to mycelial growth, virulence, asexual reproduction and cell stress response in U. virens.

Development of Generic Immuno-Magnetic Bead-Based Enzyme-Linked Immunoassay for Ustiloxins in Rice Coupled with Enrichment.

Ustiloxins are a group of mycotoxins produced by rice false smut pathogen. Previous studies have shown that the false smut balls contain six types of ustiloxins, and these toxins are toxic to living organisms. Thus, immunoassay for on-site monitoring of ustiloxins in rice is urgently required. The current immunoassays are only for detecting single ustiloxin, and they cannot meet the demand for synchronous and rapid detection of the group toxins. Therefore, this study designed and synthesized a generic antigen with ustiloxin G as material based on the common structure of the mycotoxins. Ustiloxin G was conjugated to two carrier proteins including bovine serum albumin (BSA) and ovalbvmin (OVA) by carbon diimide method. The mice were immunized with ustiloxin-G-BSA to generate the antibody serum, which was further purified to obtain the generic antibody against ustiloxins. The conjugated ustiloxin G-OVA and generic antibodies were used for establishing the enzyme-linked immunosorbent assay (ELISA) for ustiloxin detection and optimizing experiment conditions. The characterization of the antibody showed that the semi-inhibitory concentrations (IC50) of ustiloxin A, B, and G were 0.53, 0.34, and 0.06 µg/mL, respectively, and that their corresponding cross-reactivities were 11.9%, 18.4%, and 100%, respectively. To increase ELISA detection efficiency, generic antibody was combined with magnetic beads to obtain sensitive and class-specific immune-magnetic beads. Based on these immuno-magnetic beads, a high-efficiency enzyme-linked immunoassay method was developed for ustiloxin detection, whose sensitivity to ustiloxin A, B, and G was improved to 0.15 µg/mL, 0.14 µg/mL, and 0.04 µg/mL, respectively. The method accuracy was evaluated by spiking ustiloxin G as standard, and the spiked samples were tested by the immune-magnetic bead-based ELISA. The result showed the ustiloxin G recoveries ranged from 101.9% to 116.4% and were accepted by a standard HPLC method, indicating that our developed method would be promising for on-site monitoring of ustiloxins in rice.

Effect of Chemical Seed Treatment on Rice False Smut Control in Field.

Rice false smut, caused by the pathogen Ustilaginoidea virens, is a severe emerging disease in China. It affects not only the quality of rice but also yields of rice production. To make clear the effect of chemical seed treatment on the rice false smut control in fields, during 2014 to 2017, four fungicides with different modes of action were used to treat rice seeds contaminated by false smut balls. In rice-growing seasons, samples of rice tissues were taken for detection of U. virens by using a specific nested PCR method at different rice-growing stages. In addition, the occurrence of rice false smut was investigated at maturation stage. Results showed that U. virens in plant tissues decreased significantly at the seedling stage upon chemical seed treatment. Four chemical treatments decreased the detection rate significantly (P < 0.01) compared with the water treatment, but no significant difference was observed among four chemical treatments. However, the detection rate did not decease significantly at the tillering and booting stages. Similarly, the final occurrence of rice false smut did not show significant difference between each chemical and water treatment. These results suggested that chemical seed treatment had only limited efficacy in preventing occurrence of rice false smut; application of fungicides at the booting stage or integrated use of fungicides and agricultural practices might give a better control for this disease.

Extraction and purification of ustiloxin A from rice false smut balls by a combination of macroporous resin and high-speed countercurrent chromatography

Rice false smut is an emerging plant disease worldwide. Ustiloxin A (UstA) is the major mycotoxin found in rice false smut balls, which are fungal colonies in rice florets. In this study, a new method consisting of macroporous resin column chromatography and high-speed countercurrent chromatography (HSCCC) was developed for UstA separation. UstA was extracted by a 3.81% HCOOH solution and adsorbed by XAD-4 resin. UstA was then eluted by a 40% methanol solution supplemented with 0.1% trifluoroacetic acid (TFA). Further purification was achieved by HSCCC using a two-phase solvent system consisting of n-butanol/TFA/H2O (1/0.05/1, v/v/v). Under the optimized conditions, 225 mg of UstA was obtained with a purity of 97.39% in a single run, with a final recovery of 65.2%. An inhibitory effect on seed germination of wheat and maize caused by UstA was observed in a preliminary phytotoxicity assay.Graphical abstract

Rice false smut detection based on faster R-CNN

&lt;span lang="EN-IN"&gt;Rice false smut is one of the most dangerous diseases in rice at the ripening phase caused by Ustilaginoidea Virens. It is one of the most important grain diseases in rice production worldwide. Its epidemics not only lead to yield loss but also reduce grain quality because of multiple mycotoxins generated by the causative pathogen. The pathogen infects developing spikelets and specifically converts individual grain into rice false smut ball. Rice false smut balls seem to be randomly formed in some grains on a panicle of a plant in the paddy field. In this study, we suggest a novel approach for the detection of rice false smut based on faster R-CNN. The process of faster R-CNN comprises regional proposal generation and object detection. The both tasks are done in same convolutional network. Because of such design it is faster for object detection. The faster R-CNN is able to detect the RFS using rectangular labelling from on-field images. The proposed approach is the initial steps to make a prototype for the automatic detection of RFS.&lt;/span&gt;

Ustiloxin A is Produced Early in Experimental Ustilaginoidea virens Infection and Affects Transcription in Rice.

Ustiloxin is a kind of 13-membered cyclic peptides found in mature rice false smut generated by Ustilaginoidea virens infecting rice spikelet. So far, six kinds of ustiloxins have been identified from false smut balls (FSBs) in which ustiloxin A is the main component. The toxins can not only inhibit the growth of rice, wheat, and corn, but also poison people and animals. However, so far, there have been few studies of the content of ustiloxin except that in mature FSB. The effect of ustiloxins on the process of infection has not been clarified. In this study, the technique of artificial inoculation coupled with UPLC-ESI-MS was introduced to investigate the content of ustiloxins in the course of infection. The initial formation time of ustiloxin A, B, C, D, F, and G was no later than 5, 5, 9, 7, 7, and 9days post inoculation (dpi) prior to FSB's formation, respectively. The content of ustiloxin A per spikelet was increased rapidly from 6.0ng at 5 dpi to 14,157.1ng at 25 dpi. Meanwhile, the content of ustiloxin A per dry weight (DW) of the FSBs also peaked at 1321.2μg/g at 25 dpi. Interestingly,both the contents of ustiloxin A per dry weight and per spikelet were significantly reduced from 25 to 30 dpi. Transcriptome sequencing revealed that a total of 146 transcripts (103 upregulated and 43 downregulated) were significantly changed in rice spikelets after 3-h acute exposure to 100ng ustiloxin A. In addition, several of the significantly altered genes were validated by RT-qPCR.

The false smut pathogen Ustilaginoidea virens requires rice stamens for false smut ball formation.

SummaryRice false smut has emerged as a serious grain disease in rice production worldwide. The disease is characterized by the transformation of individual rice florets into false smut balls, which is caused by the fungal pathogen Ustilaginoidea virens. To date, little is known about the host factors required for false smut ball formation by U. virens. In this study, we identified histological determinants for the formation of false smut balls by inoculating U. virens into rice floral mutants defective with respect to individual floral parts. The results showed that U. virens could form mature false smut balls in rice floral mutants with defective pistils, but failed to develop false smut balls in the superwoman mutant lacking stamens, identifying that U. virens requires rice stamens to complete its infection cycle. Comparative transcriptome analysis indicated a list of candidate host genes that may facilitate nutrient acquisition by U. virens from the rice stamens, such as SWEET11, SWEET14 and SUT5, and genes involved in the biosynthesis of trehalose and raffinose family sugars. These data pinpoint rice stamens as the key target organ of U. virens infection and provide a valuable starting point for dissecting the molecular mechanism of false smut ball formation.

Application of calcined lime with simeconazole suppresses rice false smut disease

Rice false smut caused by Villosiclava virens is a devastating disease of rice. Because the primary infection source is chlamydospores in the soil, the effect of mixing calcined lime into paddy soil in combination with spraying the fungicide simeconazole on submerged soil before rice heading on the incidence of rice false smut was tested in 2016 and 2017. The calcined lime decreased the number of false smut balls, and additional spraying of simeconazole on paddy fields further decreased the incidence. Our findings suggest that calcined lime may be decreased the number of chlamydospores in field soil, and calcined lime applied with simeconazole is effective for controlling the disease.

Acute exposure to ustiloxin A affects growth and development of early life zebrafish, Danio rerio

Ustiloxin A is a cyclopeptide mycotoxin originally isolated from rice false smut balls (FSBs) that formed in rice spikelets infected by the fungal pathogen Ustilaginoidea virens. Studies have shown that ustiloxin A was toxic to animals, but the toxicological evidence is still lacking. To reveal the negative influence of ustiloxin A on model organism, zebrafish were selected and exposed to ustiloxin A at concentrations of 0, 0.25, 2.5 or 25 μM from 2 h post-fertilization (hpf) to 144 hpf. The hatching rates of embryos in the 25 μM exposure group was 12.85% less than the control group at 96 hpf. Meanwhile, exposure to 0.25, 2.5 or 25 μM ustiloxin A resulted in a distinct dose-dependent increase in mortality rate of embryos at 96 hpf. We also found that exposed to ustiloxin A could cause some other damages on zebrafish larvae, such as growth delay and increased heart rate. In addition, the athletic behavior of zebrafish larvae exposed to ustiloxin A at 25 μM was dramatically different with that of control. Transcriptome sequencing showed that abundances of 339 transcripts (125 up-regulated and 214 down-regulated) were significantly altered in larvae exposed to 25 μM of ustiloxin A. Several of the crucial genes were validated by RT-qPCR. This is the first report on the toxicologic study of ustiloxins against model organism zebrafish. Results suggested that ustiloxins have become a potential danger for food security.

A core effector UV_1261 promotes Ustilaginoidea virens infection via spatiotemporally suppressing plant defense

False smut is a destructive grain disease of rice worldwide, characterized by false smut balls formed in rice flowers. Here we identified a small secreted protein UV_1261 contributing to virulence of Ustilaginoidea virens, the causal agent of this disease. The sequence of UV_1261 was highly conserved among isolates of U. virens and absent in other fungi. UV_1261 encodes a protein targeted to plant chloroplasts. Its expression exhibited a bimodal pattern during pathogenesis. Ectopic expression of UV_1261 in Nicotiana benthamiana and Arabidopsis led to suppression of flg22-induced ROS burst, callose deposition, and expression of defense-related genes, as well as enhanced susceptibility to powdery mildew in Arabidopsis. Down-regulation of UV_1261 via exogenous siRNA treatment resulted in reduced number of false smut balls. Consistently, stably knocking-down UV_1261 caused less number of false smut balls associated with higher expression of defense-related genes in rice flower. Taken together, our data demonstrate that UV_1261 is a core effector of U. virens essential for virulence and suppressing defense in rice flower, and thus may serve as a potential molecular target for controlling rice false smut disease.

Isolation, identification, and characterization of Ustilaginoidea virens from rice false smut balls with high ustilotoxin production potential.

Ustilaginoidea (U.) virens grows on rice grains and leads to significant rice yield losses in most of the major rice producing areas. Meanwhile, ustiloxins produced by U. virens are a serious hazard to human health and ecological safety of farmlands. The other key point is that ustiloxins have been regarded as a novel resource with their potential in the treatment of cancers. There is no better way to extract ustiloxins than from pure culture of the high ustilotoxin-producing strains. U. virens has become a key research organism. However, due to the presence of some interference components, it is a certain difficulty in the successful isolation of the strain from the false smut balls. We present here a detailed study based on the separation, screening and identification of high ustiloxins-producing strains of U. virens. Through this study, we got a satisfactory success rate of separation and provided a good solution to the problem of separation. At the same time, this study provides quality resources for researchers interested in ustiloxins as anticancer agents.