Carbon Catabolite Repressor UvCreA is Required for Development and Pathogenicity in Ustilaginoidea virens

The rice false smut disease, caused by Ustilaginoidea virens, has emerged as a significantglobal threat to rice production. The mechanism of carbon catabolite repression plays a crucial role in theefficient utilization of carbon nutrients and enzyme regulation in the presence of complex nutritionalconditions. Although significant progress has been made in understanding carbon catabolite repression infungi such as Aspergillus nidulans and Magnaporthe oryzae, its role in U. virens remains unclear. Toaddress this knowledge gap, we identified UvCreA, a pivotal component of carbon catabolite repression,in U. virens. Our investigation revealed that UvCreA localized to the nucleus. Deletion of UvCreA resultedin decreased growth and pathogenicity in U. virens. Through RNA-seq analysis, it was found that theknockout of UvCreA led to the up-regulation of 514 genes and down-regulation of 640 genes. Moreover,UvCreA was found to be involved in the transcriptional regulation of pathogenic genes and genesassociated with carbon metabolism in U. virens. In summary, our findings indicated that UvCreA isimportant in fungal development, virulence, and the utilization of carbon sources through transcriptionalregulation, thus making it a critical element of carbon catabolite repression.

bZIP Transcription Factor UvATF21 Mediates Vegetative Growth,Conidiation,Stress Tolerance and Is Required for Full Virulence of Rice False Smut Fungus Ustilaginoidea virens

bZIP proteins are widely distributed in eukaryotic organisms and regulate a diverse range of physiological processes.Several bZIP proteins have previously been identified in Ustilaginoidea virens.However,the biological roles of these bZIP proteins in this pathogen are still unknown.Here,one of these bZIP protein coding genes,UvATF21,was functionally characterized.Targeted deletion of UvATF21resulted in reduced conidiation and pathogenicity despite of the increased vegetative growth.The deletion mutants also significantly decreased the sensitivity to osmotic and oxidative stresses.Interestingly,deletion of UvATF21 exhibited different performances to cell wall integrity stress.These results indicated that UvATF21 played crucial roles in vegetative growth,conidiation,stress response,and full virulence in U.virens.

In Vitro Antifungal Activity of Dihydrochelerythrine and Proteomic Analysis in Ustilaginoidea virens

Dihydrochelerythrine(DHCHE)is an isoquinoline compound,which has distinct antifungal activity and can induce apoptosis.The antifungal activity of DHCHE against five rice pathogenic fungi was studied in vitro.At the concentration of 7.5 mg/L,DHCHE exhibited the highest efficacy among tested compounds in inhibiting mycelium growth,with an inhibition rate of 68.8%in Ustilaginoidea virens,which was approximately 2.4 times of that of validamycin(28.7%).After exposure to DHCHE,transmission electron micrographs revealed spores showed incomplete organelles,malformed cell walls and nuclear membranes,as well as irregular lipid spheres.Reactive oxygen species accumulation in treated spores was markedly higher than that in control spores.DHCHE induced cell damage increased in a dose-dependent manner,as indicated by the decrease in mitochondrial membrane potential and initiation of apoptosis.The differences of expression levels of Fip1,ACP1,PMS2 and COX13 that are important for oxidative phosphorylation and mismatch repair pathway were significant,which may be some of the reasons for the induction of apoptosis in DHCHE-treated U.virens.The protein levels of Fip1,ACP1,PMS2 and COX13 agreed with protein fold change ratio from parallel reaction monitoring Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes pathway of differentially expressed proteins were further analyzed.These findings will help to elucidate the mechanisms associated with antifungal and pro-apoptotic effects of DHCHE on U.virens,thereby aiding the potential development of novel pesticides.

Resistance of Major and Backup Rice Cultivars against Ustilaginoidea virens in Anhui Province

[Objective] The aim was to study resistance of major and backup rice cul- tivars against Ustilaginoidea virens. [Method] Experiments and surveys were made on resistance of seventeen backup rice cultivars and some major cultivars in Anhui Province to identify resistance of different rice cultivars. [Result] Yanjing No.9, 80You 226, Tianxie No.l, A01 Xian, Lvjingnuo No.6 were moderate resistant; A03 Xian and Yangjing 636 were susceptible and the rest ten showed moderate susceptibility. Based on surveys on major rice cultivars, most of Liangyou rice series are suscept- able and novel Liangyou Xiang 4 enjoys resistance to certain extent. [Conclusion] The research provided references for resistance of rice against diseases in Anhui Province.

Biological Activity of Several Fungicides against Ustilaginoidea virens

[Objective] This study aims to screen for the high effective fungicides which could significantly decrease the disease incidence and disease index of rice false smut. [Method] The inhibitory activities of the fungicide against mycelial growth of Ustilaginoidea virens were measured to in vitro evaluate the ECho values. And 17 fungicides were sprayed to evaluate the efficacy and effect of the fungicides tested in the field trials on the rice characters, [Result] The results showed that epoxicona- zole, difenoconazole, propiconazole and procloraz exhibited high inhibitory activity against mycelial growth of Ustilaginoidea virens with the ECso values 0.04, 0.07, 0.12 and 0.11 pg/ml, respectively. The results of field trials showed that the efficacy of Wen- quning, and fungicides such as difenoconazole, prochloraz, propiconazole, epoxi- conazole and their mixtures in controlling rice false smut were all 70% or more. [Conclusion] The 17 tested fungicides behaved efficacy in controlling rice false smut and did not cause drug injury on leaves and grains of rice plants, sprayed when flag leaves of rice fully expanded.

Genome-Wide Identification of Zn_(2)Cys_(6 ) Class Fungal-Specific Transcription Factors(ZnFTFs)and Functional Analysis of UvZnFTFI in Ustilaginoidea virens

Transcription factors(TFs)orchestrate the regulation of cellular gene expression and thereby determine cell functionality.In this study,we analyzed the distribution of TFs containing domains,which named as ZnFTFs,both in ascomycete and basidiomycete fungi.We found that ZnFTFs were widely distributed in these fungal species,but there was more expansion of the ZnFTF class in Ascomycota than Basidiomycota.We identified 40 ZnFTFs in Ustilaginoidea virens,and demonstrated the involvement of UvZnFTF1 in vegetative growth,conidiation,pigment biosynthesis and pathogenicity.RNA-Seq analysis suggested that UvZnFTF1 may regulate different nutrient metabolism pathways,the production of secondary metabolites,and the expression of pathogen-host interaction genes and secreted protein-encodi ng genes.Analysis of the distributi on of differe nt fungal TFs in U.virens further dem on strated that UvZnFTFs make up a large TF family and may play essential biological roles in U.virens.

UvSMEK1,a Suppressor of MEK Null,Regulates Pathogenicity,Conidiation and Conidial Germination in Rice False Smut Fungus Ustilaginoidea virens

Rice false smut,which is caused by Ustilaginoidea virens,is an emerging disease of rice spikelets in rice-growing areas worldwide.However,the infection mechanism of U.virens on rice spikelets is still unclear.Here,we characterized a suppressor of mitogen-activated protein kinase kinase or ERK kinase(MEK)null(UvSMEKI)in U.virens that is conserved among filamentous fungi.Compared with wild type U.virens strain P-1,UvSMEKI deletion mutants were defective in pathogenicity and conidial germination.In addition,conidiation of UvSMEKI deletion mutants was significantly reduced on yeast extract tryptone(YT)plates,but inc「eased in YT broth compared with the wild type.Compared with UvSMEKI expression level during the vegetative mycelia and conidiation stages,UvSMEKI dramatically increased during infection of rice florets.Surprisingly,the UvSMEKI deletion mutants exhibited higher tolerance to H_(2)O_(2) and NaCl.In summary,presented evidence suggested that UvSMEKI positively regulated pathogenicity,conidial germination and conidiation in YT broth,and negatively regulated conidiation on YT medium and tolerance to oxidative and osmotic stresses.The results enhanee our understanding of the regulatory mechanism of pathogenicity of U.virens,and present a potential molecular target for blocking rice infection by U.virens.

UvWhi2 Is Required for Stress Response and Pathogenicity in Ustilaginoidea virens

Some stress response-related genes have been identified in Ustilaginoidea virens,but it is not clear whether and how defects of stress responses affect the pathogenesis processes of U.virens.In this study,we identified a general stress response factor UvWHI2 as a homolog of Saccharomyces cerevisiae Whi2 in U.virens.The relative expression level of Uv Whi2 was significantly up-regulated during infection,suggesting that UvWHI2 may be involved in pathogenesis.Furthermore,knockout of Uv Whi2 showed decreased mycelial growth,increased conidiation in the potato sucrose medium and a defect in pathogenicity.In addition,the RNA-Seq and phenotypic analysis showed that UvWHI2 was involved in response to oxidative,hyperosmotic,cell wall stress and nutrient limitation.Further studies revealed that the defects of stress responses of the?Uvwhi2 mutant affected the formation of secondary spores on the nutrient limited surface and the rice surface,resulting in a significant reduction of pathogenicity of U.virens.Our results suggest that UvWHI2 is necessary for fungal growth,stress responses and the formation of secondary spores in U.virens.In addition,the defects of stress responses can affect the formation of secondary spores on the rice surface,and then compromise the pathogenicity of U.virens.

Field Efficacy Trial and Optimum Control Period of Rice False Smut(Ustilaginoidea virens) in Single Cropping Middle-late Rice

[Objective]The paper was to study optimum chemical agents and control periods against rice false smut( Ustilaginoidea virens) in single cropping middle-late rice. [Method]Taking chemical agents( triadimefon,armure,Jinggangmycin) as main treatments and spraying periods( within 7 d before initial heading stage,within 7 d before initial heading stage + initial heading stage,initial heading stage) as assisted treatments,the field efficacy trial and optimum control period of U. virens in single cropping middle-late rice were studied using Huanghuazhan and Jinnongsimiao as experimental materials in 2013. [Result] Three chemical agents,triadimefon,armure,Jinggangmycin,extremely reduced diseased panicle rate and disease index of U. virens,but there was no significant difference among three chemical agents. Spraying period did not have significant impact on diseased panicle rate and disease index of U. virens,whereas spraying within7 d before initial heading stage and during initial heading stage had relatively good control effect. Yield increased significantly after application of three chemical agents,and armure led to greater increase. The reason for yield increase was that seed setting rate was significantly increased,and the number of filled grains per panicle was significantly increased. Although spraying period influenced yield,the difference was not significant. [Conclusion] Triadimefon,armure and Jinggangmycin had good control effects against U. virens in single cropping middle-late rice,of which armure had better control effects. Spraying chemical agents increased seed setting rate,and further increased the number of filled grains per panicle and yield. Spraying within 7 d before initial heading stage and during initial heading stage had relatively better control effect against U. virens.

Isolation Technology of Single Spore and Optimization of Conidia Culture Condition for Ustilaginoidea virens

The single spores were isolated from chlamydospores of Ustilaginoidea virens with three different maturities by PSA. The isolated single spores were cul- tured on different media at different temperatures under natural light for inducing conidia to explore the optimum isolation technique of single spore and optimum cul- ture condition of conidia. The results showed that the successful isolating rate of single spore from yellow rice false smut balls reached 90.00%. The sporulatina quantities of isolated single spores cultured on PSD and PDB media at 22 -29 ~C （variable temperature under natural light） or 28 ℃ （constant temperature under dark condition） for 12 d were up to 6.3× 107 and 1.1× 106 spore/mL, respectively. PSA was the most effective method to isolate single spores from yellow rice false smut balls of U. virens. The optimum conidia culture condition included PSD or PDB medium, 22 -29℃ or 28℃, natural light and vibration culture. Key words Ustilaginoidea virens; Single spore isolation; Conidia; Culture condition

Preservation and Conidia Culture Technique of Ustilaginoidea virens

[Objective] This study aimed to find methods suitable for the preservation and conidia production of Ustilaginoidea virens. [Method] Six different preservation methods were tested by checking at three month interval for the viability and sporulation, and seven liquid media of different composition were tested by shaking cul- ture at 150 r/min. [Result] Among the six methods studied, the periodic transfer and paraffin oil overlay were suitable, and the fungus could survive more than 12 months. The conidia concentration went up to 7.25×10^7 conidia/ml after 9 d in the PSB liquid medium, indicating that PSB was the best medium for conidia production. [Conclusion] This study provides a theoretical basis for the preservation and conidia production of U. virens.

Rapid detection of the rice false smut fungus Ustilaginoidea virens by lateral flow strip-based recombinase polymerase amplification assay

Rice false smut,caused by Ustilaginoidea virens,is a devastating disease that greatly reduces rice yield and quality.However,controlling rice false smut disease is challenging due to the unique infection mode of U.virens.Therefore,there is a need for early diagnosis and monitoring techniques to prevent the spread of this disease.Lateral flow strip-based recombinase polymerase amplification(LF-RPA)overcomes the limitations of current U.virens detection technologies,which are time-consuming,require delicate equipment,and have a high false-positive rate.In this study,we used a comparative genomics approach to identify Uv_3611,a specific gene of U.virens,as the target for the LF-RPA assay.The designed primers and probe efffectively detected the genomic DNA(gDNA)of U.virens and demonstrated no cross-reactivity with related pathogens.Under optimal conditions,the LF-RPA assay demonstrated a sensitivity of 10 pg of U.virens gDNA.Additionally,by incorporating a simplified PEG-NaOH method for plant DNA extraction,the LF-RPA assay enabled the detection of U.virens in rice spikelets within 30 min,without the need for specialized equipment.Furthermore,the LF-RPA assay successfully detected U.virens in naturally infected rice and seed samples in the field.Therefore,the LF-RPA assay is sensitive,efficient,and convenient,and could be developed as a kit for monitoring rice false smut disease in the field.

Comprehensive identification of lysine 2-hydroxyisobutyrylated proteins in Ustilaginoidea virens reveals the involvement of lysine 2-hydroxyisobutyrylation in fungal virulence

Lysine 2-hydroxyisobutyrylation(Khib) is a newly identified post-translational modification(PTM) that plays important roles in transcription and cell proliferation in eukaryotes. However, its function remains unknown in phytopathogenic fungi. Here,we performed a comprehensive assessment of Khibin the rice false smut fungus Ustilaginoidea virens, using Tandem Mass Tag(TMT)-based quantitative proteomics approach. A total of 3 426 Khibsites were identified in 977 proteins, sugg esting that Khibis a common and complex PTM in U. virens. Our data demonstrated that the2-hydroxyisobutyrylated proteins are involved in diverse biological processes. Network analysis of the modified proteins revealed a highly interconnected protein network that included many well-studied virulence factors. We confirmed that the Zn-binding reduced potassium dependency3-type histone deacetylase(UvRpd3) is a major enzyme that removes 2-hydroxyisobutyrylation and acetylation in U. virens. Notably, mutations of Khib sites in the mitogen-activated protein kinase(MAPK)UvSlt2 significantly reduced fungal virulence and decreased the enzymatic activity of UvSlt2. Molecular dynamics simulations demonstrated that 2-hydroxyisobutyrylation in UvSlt2 increased the hydrophobic solvent-accessible surface area and thereby affected binding between the UvSlt2 enzyme and its substrates. Our findings thus establish Khibas a major post-translational modification in U. virens and point to an important role for Khibin the virulence of this phytopathogenic fungus.

Ustilaginoidea virens modulates lysine 2-hydroxyisobutyrylation in rice flowers during infection

The post-translational modification lysine 2-hydroxyisobutyrylation(K_(hib))plays an important role in gene transcription,metabolism,and enzymatic activity.K_(hib)sites have been identified in rice(Oryza sativa).However,the K_(hib)status of proteins in rice flowers during pathogen infection remains unclear.Here,we report a comprehensive identification of K_(hib)-modified proteins in rice flowers,and the changes in these proteins during infection with the fungal pathogen Ustilaginoidea virens.By using a tandem mass tag-based quantitative proteomics approach,we identified 2,891 K_(hib)sites on 964 proteins in rice flowers.Our data demonstrated that 2-hydroxyisobutyrylated proteins are involved in diverse biological processes.K_(hib)levels were substantially reduced upon infection with U.virens.Chromatin immunoprecipitation polymerase chain reaction(PCR)and reverse transcription quantitative PCR analyses revealed that histone K_(hib)is involved in the expression of disease-resistance genes.More importantly,most quantified sites on core histones H3 were downregulated upon U.virens infection.In addition,the histone deacetylases HDA705,HDA716,SRT1,and SRT2 are involved in the removal of K_(hib)marks in rice.HDA705 was further confirmed to negatively regulate rice disease resistance to pathogens U.virens,Magnaporthe oryzae,and Xanthomonas oryzae pv.oryzae(Xoo).Our data suggest that U.virens could modulate K_(hib)in rice flowers during infection.

Effect of temperature on the development of sclerotia in Villosiclava virens

The sclerotia of Villosiclava virens were found commonly in high altitude and the temperate regions, where the temperatures are relatively low in rice filling stage. To make sure if low temperature induce the sclerotial formation in V. virens, the inoculated rice panicles in laboratory and the diseased rice panicles cut from paddy fields were treated under different temperatures. The results showed that 3 days of night temperature at 15°C were enough to induce the sclerotial formation. The low temperature was much more effective for young balls with intact membranes. After appearance of chlamydospores on the ball surfaces, the sclerotium could not differentiate anymore. The sclerotia began to differentiate below the chlamydospore layer and gradually grew onto the ball surfaces. This suggests that low temperature in the early development stage of false smut balls is an important factor to induce the sclerotial differentiation, and rice cultivars with long growth periods are able to produce more sclerotium-bearing balls, which will produce mass of spores in paddy field in the coming year.

Putative Phosphatase UvPsr1 Is Required for Mycelial Growth, Conidiation, Stress Response and Pathogenicity in Ustilaginonidea virens

Ustilaginoidea virens is the causal agent of rice false smut,which can be a highly destructive disease of rice.The plasma membrane phosphatase Psr1 proteins,which act as a regulator of the salinity stress response in yeast,are widely distributed across fungi,but their functional characterization is sketchy.In this study,we characterized the functions of Psr1 protein,UvPsr1,in U.virens.Analyses of the AUvpsr1 and its complementation strain showed that UvPsr1 is required for normal mycelial growth,conidiation and tolerance to oxidative,osmotic and cell wall stresses.When rice panicles were inoculated with the AUvpsr1 strains,no symptoms of false smut disease developed,showing that UvPSR1 also contributes to the pathogenicity of the fungus.The deletion mutant of UvPSR1 also appeared to produce a smaller titer of toxic compounds able to inhibit elongation of the germinated seeds.In conclusion,our results indicated that UvPsr1 is a new pathogenic factor of U.virens.

Integrated Approach to Control False Smut in Hybrid Rice in Sichuan Province, China

Severe epidemic of false smut, caused by Ustilaginoidea virens （Cooke） Takahashi （teleomorph Villosiclava virens） has been reported in different parts of Asia and America. Different fungicides or bio-control agents against false smut were applied at different times before heading on a susceptible rice variety Pu-6. A control efficiency as high as 91.92% was resulted from spraying 2.5% Wenquning, a suspension of Bacillus subtilis in solution of validamycin with 4.5 L/hm2 at 6 d before heading. Among the 186 hybrid rice screened in 2010, low significant correlations between the dates of full heading, rates of infected plants and panicles as well as the number of infected florets were found, with the correlation coefficients ranging from 0.2331 to 0.5212. However, significant difference in susceptibility coefficients was also found between the varieties which had the same dates of full heading. In the plot experiments to compare the susceptibility in 2011, the average rates of infected panicles of Yixiangyou 2168, Chuanxiangyou 3, Dexiang 4103, Yixiangyou 2115, Nei5you 317, Yangxinyou 1 were significantly lower than those of the control varieties Gangyou 725 and Gangyou 188 at the disease nursery located at Qionglai, Sichuan Province, China. When Neixiangyou 8156 and Nei5you 317 were sprayed with 2.5% Wenquning at 4.5 L/hm2 for two times at 6 d before and 1 d after heading, respectively, the control efficiencies of Nei5you 317 and Neixiangyou 8156 were respectively 100% and 82.24% compared to that of Gangyou 725. Satisfactory control effects had also obtained by single spray of 2.5% Wenquning at 4.5 L/hm2 at 5-6 d before heading. Therefore, less susceptible hybrid rice in combination with spraying Wenquning at 5-6 d before heading was suggested for the disease control in Sichuan Province, China.

Evaluation of 0.3% Agricultural Antibiotic 702 AS against Three Pathogenic Fungi of Rice

Abstractsolution （AS） against Rhizocto- nia solani, Magnaporthe grisea and Ustilaginoidea virens. [ Method] The toxicity effect of 0.3% agricultural antibiotic 702 AS against R., solani, M. grisea and U. virens were studied by mycelial growth rate method and mycelial wet weight method. [ Result ] The EC50 and EC50 values of agricultural antibiotics 702 AS to R. solani, M. grisea and U. virens in vitro condition were 4.16 and 16.17 mg/L, 16.05 and 41.85 mg/L, 111.2 and 389.0 mg/L, respectively. Agricultural antibi- otics 702 AS had good inhibition effect against R. solani and M. grisea and could basically achieve the effects of commercially available Jinggangmycin and kaanga- mycin, but the inhibition effect against U. v/rens was relatively weak. The preventive activity of aqueous solution was obviously higher than treatment activity. [Conclusion] 0.3% Agricultural antibiotic 702 AS is considered to be a potential biological control agent against R. solani, M. grisea and U. virens, and can be used as the potential alternative pesticide of Jinggangmycin.