Co-infection of Four Novel Mycoviruses from Three Lineages Confers Hypovirulence on Phytopathogenic Fungus Ustilaginoidea virens.

Rice false smut caused by Ustilaginoidea virens has become one of the most important diseases of rice. Mycoviruses are viruses that can infect fungi with the potential to control fungal diseases. However, little is known about the biocontrol role of hypoviruses in U. virens. In this study, we revealed that the hypovirulence-associated U. virens strain Uv325 was co-infected by four novel mycoviruses from three lineages, designated Ustilaginoidea virens RNA virus 16 (UvRV16), Ustilaginoidea virens botourmiavirus virus 8 (UvBV8), Ustilaginoidea virens botourmiavirus virus 9 (UvBV9), and Ustilaginoidea virens narnavirus virus 13 (UvNV13), respectively. The U. virens strain co-infected by four mycoviruses showed slower growth rates, reduced conidial yield, and attenuated pigmentation. We demonstrated that UvRV16 was not only the major factor responsible for the hypovirulent phenotype in U. vriens, but also able to prevent U. virens to accumulate more mycotoxin, thereby weakening the inhibitory effects on rice seed germination and seedling growth. Additionally, we indicated that UvRV16 can disrupt the antiviral response of U. virens by suppressing the transcriptional expression of multiple genes involved in autophagy and RNA silencing. In conclusion, our study provided new insights into the biological control of rice false smut.

Metabolomics analysis of the effects of chelerythrine on Ustilaginoidea virens.

Rice false smut (RFS) caused by Ustilaginoidea virens is widely distributed in major rice-producing regions. Previous studies have shown that treating RFS with chelerythrine can decrease the germination of fungus spores by 86.7% and induce fungal cell apoptosis. In the present study, the effects of chelerythrine on the metabolism of U. virens explored using metabolomics and analyses of differentially accumulated metabolites and altered metabolic pathways. The top 15 metabolites in random forest analysis were significantly different between groups. In positive ion mode, purine, phenylalanine metabolism, phenylalanine, tyrosine, tryptophan biosynthesis, pyrimidine metabolism, and nitrogen metabolism were dominant. Alanine, aspartate, glutamate metabolism, and phenylalanine metabolism were enriched in negative ion mode. Differentially expressed genes and altered metabolic pathways of U. virens were effected by chelerythrine. The findings support future research on the prevention and treatment of RFS by chelerythrine and provide a theoretical basis for targeted drug delivery.

Exploring the potential of Bacillus for crop productivity and sustainable solution for combating rice false smut disease.

Rice false smut, which is caused by the soil-borne fungal pathogen Ustilaginoidea virens (U. virens), is one of the most threatening diseases in most of the rice-growing countries including India that causes 0.5-75% yield loss, low seed germination, and a reduction in seed quality. The assessment of yield loss helps to understand the relevance of disease severity and facilitates the implementation of appropriate management strategies. This study aimed to mitigate biotic stress in rice by employing a rhizobacterial-based bioformulation, which possesses diverse capabilities as both a plant growth promoter and a biocontrol agent against U. virens. Rhizobacteria were isolated from the soil of the rice rhizospheres from the healthy plant of the false smut affected zone. Furthermore, they were identified as Bacillus strains: B. subtilis (BR_4), B. licheniformis (BU_7), B. licheniformis (BU_8), and B. vallismortis (KU_7) via sequencing. Isolates were screened for their biocontrol potential against U. virens under in vitro conditions. The antagonistic study revealed that B. vallismortis (KU_7) inhibited U. virens the most (44.6%), followed by B. subtilis BR_4 (41.4%), B. licheniformis BU_7 (39.8%), and B. licheniformis BU_8 (43.5%). Various biochemical and plant growth promoting attributes, such as phosphate and Zn solubilization, IAA, ammonium, siderophore, and chitinase production, were also investigated for all the selected isolates. Furthermore, the potential of the isolates was tested in both in vitro and field conditions by employing talc-based bioformulation through bio-priming and root treatment. The application of bioformulation revealed a 20% decrease in disease incidence in plants treated with B. vallismortis (KU_7), a 60.5% increase in the biological yield, and a 45% increase in the grain yield. This eco-friendly approach not only controlled the disease but also improved the grain quality and reduced the chaffiness.

Conjunctive Analysis of BSA-Seq and SSR Markers Unveil the Candidate Genes for Resistance to Rice False Smut.

Rice false smut (RFS) caused by the fungus Ustilaginoidea virens (Cook) leads to serious yield losses in rice. Identification of the gene or quantitative trait loci (QTLs) is crucial to resistance breeding and mitigation of RFS damage. In this study, we crossed a resistant variety, IR77298-14-1-2::IRGC117374-1, with a susceptible indica cultivar, 9311, and evaluated recombinant inbred lines in a greenhouse. The genetic analysis showed that the RFS resistance of IR77298-14-1-2::IRGC117374-1 was controlled by multiple recessive loci. We identified a novel QTL, qRFS12.01, for RFS resistance in IR77298-14-1-2::IRGC117374-1 by combining bulked segregant analysis with whole genome resequencing (BSA-seq) and simple sequence repeat (SSR) marker mapping approaches. The phenotypic effect of qRFS12.01 on RFS resistance reached 28.74%, suggesting that SSR markers linked to qRFS12.01 are valuable for marker-assisted breeding of RFS resistance in rice. The prediction of putative candidate genes within qRFS12.01 revealed five disease resistance proteins containing NB-ARC domains. In conclusion, our findings provide a new rice chromosome region carrying genes/QTLs for resistance to RFS.

Ustilaginoidea virens-secreted effector Uv1809 suppresses rice immunity by enhancing OsSRT2-mediated histone deacetylation.

Rice false smut caused by Ustilaginoidea virens is a devastating rice (Oryza sativa) disease worldwide. However, the molecular mechanisms underlying U. virens-rice interactions are largely unknown. In this study, we identified a secreted protein, Uv1809, as a key virulence factor. Heterologous expression of Uv1809 in rice enhanced susceptibility to rice false smut and bacterial blight. Host-induced gene silencing of Uv1809 in rice enhanced resistance to U. virens, suggesting that Uv1809 inhibits rice immunity and promotes infection by U. virens. Uv1809 suppresses rice immunity by targeting and enhancing rice histone deacetylase OsSRT2-mediated histone deacetylation, thereby reducing H4K5ac and H4K8ac levels and interfering with the transcriptional activation of defence genes. CRISPR-Cas9 edited ossrt2 mutants showed no adverse effects in terms of growth and yield but displayed broad-spectrum resistance to rice pathogens, revealing a potentially valuable genetic resource for breeding disease resistance. Our study provides insight into defence mechanisms against plant pathogens that inactivate plant immunity at the epigenetic level.

A Genome-Wide Comparison of Rice False Smut Fungus Villosiclava virens Albino Strain LN02 Reveals the Genetic Diversity of Secondary Metabolites and the Cause of Albinism.

Rice false smut (RFS) caused by Villosiclava virens (anamorph: Ustilaginoidea virens) has become one of the most destructive fungal diseases to decrease the yield and quality of rice grains. An albino strain LN02 was isolated from the white RFS balls collected in the Liaoning Province of China in 2019. The strain LN02 was considered as a natural albino mutant of V. virens by analyzing its phenotypes, internal transcribed spacer (ITS) conserved sequence, and biosynthesis gene clusters (BGCs) for secondary metabolites. The total assembled genome of strain LN02 was 38.81 Mb, which was comprised of seven nuclear chromosomes and one mitochondrial genome with an N50 value of 6,326,845 bp and 9339 protein-encoding genes. In addition, the genome of strain LN02 encoded 19 gene clusters for biosynthesis of secondary metabolites mainly including polyketides, terpenoids and non-ribosomal peptides (NRPs). Four sorbicillinoid metabolites were isolated from the cultures of strain LN02. It was found that the polyketide synthase (PKS)-encoding gene uspks1 for ustilaginoidin biosynthesis in strain LN02 was inactivated due to the deletion of four bases in the promoter sequence of uvpks1. The normal uvpks1 complementary mutant of strain LN02 could restore the ability to synthesize ustilaginoidins. It demonstrated that deficiency of ustilaginoidin biosynthesis is the cause of albinism for RFS albino strain LN02, and V. virens should be a non-melanin-producing fungus. This study further confirmed strain LN02 as a white phenotype mutant of V. virens. The albino strain LN02 will have a great potential in the development and application of secondary metabolites. The physiological and ecological functions of ustilaginoidins in RFS fungus are needed for further investigation.

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

Introduction: 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. Methods: 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. Results: 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. Discussion: 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.

An Ustilaginoidea virens glycoside hydrolase 42 protein is an essential virulence factor and elicits plant immunity as a PAMP.

Rice false smut, caused by the ascomycete fungus Ustilaginoidea virens, which infects rice florets before heading, severely threatens rice grain yield and quality worldwide. The U. virens genome encodes a number of glycoside hydrolase (GH) proteins. So far, the functions of these GHs in U. virens are largely unknown. In this study, we identified a GH42 protein secreted by U. virens, named UvGHF1, that exhibits ß-galactosidase activity. UvGHF1 not only functions as an essential virulence factor during U. virens infection, but also serves as a pathogen-associated molecular pattern (PAMP) in Nicotiana benthamiana and rice. The PAMP activity of UvGHF1 is independent of its ß-galactosidase activity. Moreover, UvGHF1 triggers cell death in N. benthamiana in a BAK1-dependent manner. Ectopic expression of UvGHF1 in rice induces pattern-triggered immunity and enhances rice resistance to fungal and bacterial diseases. RNA-seq analysis revealed that UvGHF1 expression in rice not only activates expression of many defence-related genes encoding leucine-rich repeat receptor-like kinases and WRKY and ERF transcription factors, but also induces diterpenoid biosynthesis and phenylpropanoid biosynthesis pathways. Therefore, UvGHF1 contributes to U. virens virulence, but is also recognized by the rice surveillance system to trigger plant immunity.

Sucrose non-fermenting protein kinase gene UvSnf1 is required for virulence in Ustilaginoidea virens.

Rice false smut caused by Ustilaginoidea virens is becoming one of the most devastating diseases in rice production areas in the world. Revealing U. virens potential pathogenic mechanisms provides ideas for formulating more effective prevention and control strategies. Sucrose non-fermenting 1 (Snf1) protein kinase plays a critical role in activating transcription and suppressing gene expression, as well as in cellular response to various stresses, such as nutrient limitation. In our study, we identified the Snf1 homolog UvSnf1 and analyzed its biological functions in U. virens. The expression level of UvSnf1 was dramatically up-regulated during invasion, indicating that UvSnf1 may participate in infection. Phenotypic analyses of UvSnf1 deletion mutants revealed that UvSnf1 is necessary for hyphae growth, spore production, and virulence in U. virens. Moreover, UvSnf1 promotes U. virens to use unfavorable carbon sources when the sucrose is insufficient. In addition, deletion of UvSnf1 down-regulates the expression of the cell wall-degrading enzymes (CWDEs) genes under sucrose limitation conditions in U. virens. Further analyses showed that CWDEs (UvCut1 and UvXyp1) are not only involved in growth, spore production, and virulence but are also required for the utilization of carbon sources. In conclusion, this study demonstrates that UvSnf1 plays vital roles in virulence and carbon source utilization in U. virens, and one of the possible mechanisms is playing a role in regulating the expression of CWDE genes.

Quantitative Loop-Mediated Isothermal Amplification Detection of Ustilaginoidea virens Causing Rice False Smut.

Rice false smut caused by Ustilaginoidea virens is one of the most devastating diseases in rice worldwide, which results in serious reductions in rice quality and yield. As an airborne fungal disease, early diagnosis of rice false smut and monitoring its epidemics and distribution of its pathogens is particularly important to manage the infection. In this study, a quantitative loop-mediated isothermal amplification (q-LAMP) method for U. virens detection and quantification was developed. This method has higher sensitivity and efficiency compared to the quantitative real-time PCR (q-PCR) method. The species-specific primer that the UV-2 set used was designed based on the unique sequence of the U. virens ustiloxins biosynthetic gene (NCBI accession number: BR001221.1). The q-LAMP assay was able to detect a concentration of 6.4 spores/mL at an optimal reaction temperature of 63.4 °C within 60 min. Moreover, the q-LAMP assay could even achieve accurate quantitative detection when there were only nine spores on the tape. A linearized equation for the standard curve, y = -0.2866x + 13.829 (x is the amplification time, the spore number = 100.65y), was established for the detection and quantification of U. virens. In field detection applications, this q-LAMP method is more accurate and sensitive than traditional observation methods. Collectively, this study has established a powerful and simple monitoring tool for U. virens, which provides valuable technical support for the forecast and management of rice false smut, and a theoretical basis for precise fungicide application.

Inhibition of rice germination by ustiloxin A involves alteration in carbon metabolism and amino acid utilization.

Ustiloxins are the main mycotoxin in rice false smut, a devastating disease caused by Ustilaginoidea virens. A typical phytotoxicity of ustiloxins is strong inhibition of seed germination, but the physiological mechanism is not clear. Here, we show that the inhibition of rice germination by ustiloxin A (UA) is dose-dependent. The sugar availability in UA-treated embryo was lower while the starch residue in endosperm was higher. The transcripts and metabolites responsive to typical UA treatment were investigated. The expression of several SWEET genes responsible for sugar transport in embryo was down-regulated by UA. Glycolysis and pentose phosphate processes in embryo were transcriptionally repressed. Most of the amino acids detected in endosperm and embryo were variously decreased. Ribosomal RNAs for growth were inhibited while the secondary metabolite salicylic acid was also decreased under UA. Hence, we propose that the inhibition of seed germination by UA involves the block of sugar transport from endosperm to embryo, leading to altered carbon metabolism and amino acid utilization in rice plants. Our analysis provides a framework for understanding of the molecular mechanisms of ustiloxins on rice growth and in pathogen infection.

SPR9 encodes a 60 S ribosomal protein that modulates panicle spreading and affects resistance to false smut in rice (Oryza sativa. L).

BACKGROUND: The architecture of inflorescence in crops is a key agronomic feature determining grain yield and thus has been a major target trait of cereal domestication. RESULTS: In this study, we show that a simple spreading panicle change in rice panicle shape, controlled by the Spreading Panicle 9 (SPR9) locus, also has a significant impact on the resistance to rice false smut (RFS). Meanwhile, we mapped a novel spr9 mutant gene between markers Indel5-18 and Indel5-22 encompassing a genomic region of 43-kb with six candidate genes. Through gene prediction and cDNA sequencing, we confirmed that LOC_Os05g38520 is the target gene in the spr9 mutant, which encodes 60 S ribosomal protein L36-2. Further analysis showed that the spr9 mutant is caused by a 1 bp deletion in the first exon that resulted in premature termination. Knockout experiments showed that the SPR9 gene is responsible for the spreading panicle phenotype of the spr9 mutant. Interestingly, the spr9 mutant was found to improve resistance to RFS without affecting major agronomic traits. Taken together, our results revealed that the spr9 allele has good application prospects in rice breeding for disease resistance and panicle improvement. CONCLUSIONS: We report the map-based cloning and functional characterization of SPR9, which encodes a 60 S ribosomal protein that regulates spreading panicles and affects the resistance to false smut in rice.

The Genetic Mechanism of the Immune Response to the Rice False Smut (RFS) Fungus Ustilaginoidea virens.

Rice false smut (RFS), which is caused by Ustilaginoidea virens (U. virens), has become one of the most devastating diseases in rice-growing regions worldwide. The disease results in a significant yield loss and poses health threats to humans and animals due to producing mycotoxins. In this review, we update the understanding of the symptoms and resistance genes of RFS, as well as the genomics and effectors in U. virens. We also highlight the genetic mechanism of the immune response to RFS. Finally, we analyse and explore the identification method for RFS, breeding for resistance against the disease, and interactions between the effector proteins and resistance (R) proteins, which would be involved in the development of rice disease resistance materials for breeding programmes.

Overexpression of a beta-1,6-glucanase gene GluM in transgenic rice confers high resistance to rice blast, sheath blight and false smut.

BACKGROUND: Frequent fungal diseases tend to lead to severe losses in rice production. As a main component of the fungal cell wall, glucan plays an important role in the growth and development of fungi. Glucanase can inhibit the growth of fungi by breaking glycosidic bonds, and may be a promising target for developing rice varieties with broad-spectrum disease resistance. RESULTS: We transferred a codon-optimized ß-1,6-glucanase gene (GluM) from myxobacteria into the japonica rice variety Zhonghua11 (ZH11), and obtained a large number of individual transgenic plants with GluM overexpression. Based on molecular analysis, three single-copy homozygous lines with GluM overexpression were selected for assessment of fungal disease resistance at the T3 generation. Compared with that of the recipient cultivar ZH11, the area of rice blast lesion in transgenic rice was reduced by 82.71%; that of sheath blight lesion was decreased by 35.76%-43.67%; the sheath blight resistance in the field was enhanced by an average of 0.75 grade over 3 years; and the incidence of diseased panicles due to rice false smut was decreased by 65.79%. More importantly, there was no obvious loss of yield (without a significant effect on agronomic traits). Furthermore, plants overexpressing a ß-1,6-glucanase gene showed higher disease resistance than rice plants overexpressing a ß-1,3-glucanase gene derived from tobacco. CONCLUSION: The ß-1,6-glucanase gene GluM can confer broad-spectrum disease resistance to rice, providing an environmentally friendly alternative way to effectively manage fungal pathogens in rice production. © 2023 Society of Chemical Industry.

Combination effects of tebuconazole with Bacillus subtilis to control rice false smut and the related synergistic mechanism.

BACKGROUND: Management of rice false smut is based mainly on chemical control, which poses many safety and environmental challenges. The other option, biological control with biofungicides, does not have such problems but is not as reliable because of low systemic ability, and lower and unstable efficacy. Therefore, it is necessary to combine application of chemical fungicides and biological control agents (BCAs) and elucidate their synergistic mechanism. RESULTS: A combination of tebuconazole and a proven BCA, Bacillus subtilis H158, was evaluated for control of rice false smut. Tebuconazole at low application rates stimulated growth of B. subtilis, prolonged the effective period of B. subtilis by enhancing its persistence on the surface of rice plants, accelerated biofilm formation of the BCA to facilitate colonization, promoted induced systemic resistance in rice by regulating defense-related enzymes and genes, and reduced the natural resistance of the pathogen by suppressing the key gene for fungal resistance to tebuconazole. However, at high application rates, tebuconazole had adverse effects on these factors and showed antagonistic combination effects with B. subtilis. The combination of B. subtilis with tebuconazole at low application rates showed great synergistic effects, but at high application rates showed only antagonistic effects in field experiments over two consecutive years. CONCLUSION: The combination of B. subtilis with tebuconazole had significant synergistic effects at low application rates. The synergistic effects are the result of multiple mechanisms involved in BCA, rice and the fungal pathogen. © 2022 Society of Chemical Industry.

Activation of Ustilaginoidin Biosynthesis Gene uvpks1 in Villosiclava virens Albino Strain LN02 Influences Development, Stress Responses, and Inhibition of Rice Seed Germination.

Villosiclava virens (anamorph: Ustilaginoidea virens) is the pathogen of rice false smut (RFS), which is a destructive rice fungal disease. The albino strain LN02 is a natural white-phenotype mutant of V. virens due to its incapability to produce toxic ustilaginoidins. In this study, three strains including the normal strain P1, albino strain LN02, and complemented strain uvpks1C-1 of the LN02 strain were employed to investigate the activation of the ustilaginoidin biosynthesis gene uvpks1 in the albino strain LN02 to influence sporulation, conidia germination, pigment production, stress responses, and the inhibition of rice seed germination. The activation of the ustilaginoidin biosynthesis gene uvpks1 increased fungal tolerances to NaCl-induced osmotic stress, Congo-red-induced cell wall stress, SDS-induced cell membrane stress, and H2O2-induced oxidative stress. The activation of uvpks1 also increased sporulation, conidia germination, pigment production, and the inhibition of rice seed germination. In addition, the activation of uvpks1 was able to increase the mycelial growth of the V. virens albino strain LN02 at 23 °C and a pH from 5.5 to 7.5. The findings help in understanding the effects of the activation of uvpks1 in albino strain LN02 on development, pigment production, stress responses, and the inhibition of rice seed germination by controlling ustilaginoidin biosynthesis.

Diversity Analysis of the Rice False Smut Pathogen Ustilaginoidea virens in Southwest China.

Rice false smut caused by Ustilaginoidea virens is a destructive disease in rice cropping areas of the world. The present study is focused on the morphology, pathogenicity, mating-type loci distribution, and genetic characterization of different isolates of U. virens. A total of 221 strains of U. virens were collected from 13 rice-growing regions in southwest China. The morphological features of these strains exhibited high diversity, and the pathogenicity of the smut fungus showed significant differentiation. There was no correlation between pathogenicity and sporulation. Mating-type locus (MAT) analysis revealed that all 221 isolates comprised heterothallic and homothallic forms, wherein 204 (92.31%) and 17 (7.69%) isolates belonged to heterothallic and homothallic mating types, respectively. Among 204 strains of heterothallic mating types, 62 (28.05%) contained MAT1-1-1 idiomorphs, and 142 isolates (64.25%) had the MAT1-2-1 idiomorph. Interestingly, strains isolated from the same fungus ball had different mating types. The genetic structure of the isolates was analyzed using simple sequence repeats (SSRs) and single-nucleotide polymorphisms (SNPs). All isolates were clustered into five genetic groups. The values of Nei's gene diversity (H) and Shannon's information index (I) indicated that all strains as a group had higher genetic diversity than strains from a single geographical population. The pairwise population fixation index (FST) values also indicated significant genetic differentiation among all compared geographical populations. The analysis of molecular variation (AMOVA) indicated greater genetic variation within individual populations and less genetic variation among populations. The results showed that most of the strains were not clustered according to their geographical origin, showing the rich genetic diversity and the complex and diverse genetic background of U. virens in southwest China. These results should help to better understand the biological and genetic diversity of U. virens in southwest China and provide a theoretical basis for building effective management strategies.

UvSorA and UvSorB Involved in Sorbicillinoid Biosynthesis Contribute to Fungal Development, Stress Response and Phytotoxicity in Ustilaginoidea virens.

Ustilaginoidea virens (teleomorph: Villosiclava virens) is an important fungal pathogen that causes a devastating rice disease. It can produce mycotoxins including sorbicillinoids. The biosynthesis and biological functions of sorbicillinoids have not been reported in U. virens. In this study, we identified a sorbicillinoid biosynthetic gene cluster in which two polyketide synthase genes UvSorA and UvSorB were responsible for sorbicillinoid biosynthesis in U. virens. In ∆UvSorA and ∆UvSorB mutants, the mycelial growth, sporulation and hyphal hydrophobicity were increased dramatically, while the resistances to osmotic pressure, metal cations, and fungicides were reduced. Both phytotoxic activity of rice germinated seeds and cell wall integrity were also reduced. Furthermore, mycelia and cell walls of ∆UvSorA and ∆UvSorB mutants showed alterations of microscopic and submicroscopic structures. In addition, feeding experiment showed that sorbicillinoids could restore mycelial growth, sporulation, and cell wall integrity in ∆UvSorA and ∆UvSorB mutants. The results demonstrated that both UvSorA and UvSorB were responsible for sorbicillinoid biosynthesis in U. virens, and contributed to development (mycelial growth, sporulation, and cell wall integrity), stress responses, and phytotoxicity through sorbicillinoid mediation. It provides an insight into further investigation of biological functions and biosynthesis of sorbicillinoids.

A Nanobody-Based Immunoassay for Detection of Ustilaginoidins in Rice Samples.

Ustilaginoidins are a class of bis-naphtho-γ-pyrone mycotoxins produced by the pathogen Villosiclava virens of rice false smut, which has recently become one of the most devastating diseases in rice-growing regions worldwide. In this research, the nanobody phage display library was established after an alpaca was immunized with the hemiustilaginoidin F-hapten coupled with bovine serum albumin (BSA). Heterologous antigen selection and combing trypsin with competition alternant elution methods were performed for nanobody screening. Two nanobodies, namely, Nb-B15 and Nb-C21, were selected for the establishment of indirect competitive enzyme-linked immunosorbent assays (ic-ELISAs). For Nb-B15 and Nb-C21, their IC50 values were 11.86 µg/mL and 11.22 µg/mL, and the detection ranges were at 3.41-19.98 µg/mL and 1.17-32.13 µg/mL, respectively. Two nanobodies had a broad spectrum to quantify the contents of total ustilaginoidins in rice samples according to cross-reactivity. The recognition mechanisms of Nb-B15 and Nb-C21 against ustilaginoidin A were elucidated by molecular modeling and docking. The key amino acid sites for the binding of Nb-B15 or Nb-C21 to ustilaginoidin A were mainly located in the FR1 and CDR1 regions. As Nb-B15 was superior to Nb-C21 in the aspects of protein expression, ELISA titer, and tolerance to organic solvents, it was selected for application in the detection of actual contaminated rice samples. The total ustilaginoidin contents of rice samples were analyzed by Nb-B15-based ic-ELISA and HPLC-DAD, between which the results were found to be consistent. The developed immunoassay based on the nanobody from the alpaca can be employed as a rapid and effective method for detection of total utilaginoidins in contaminated rice samples.

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.