Rice Kernel Smut Research Articles

Genome sequencing, phylogenomics, and population analyses of Tilletia, with recognition of one common bunt species, T. caries (synonym T. laevis), distinct from dwarf bunt, T. controversa

ABSTRACT Some species of Tilletia are responsible for diseases in economically important crops, such as wheat and rice. In this study, we sequenced, assembled, and annotated 22 new genomes for Tilletia, with a focus on species causing dwarf bunt (DB; T. controversa), common bunt (CB; T. caries and T. laevis), and rice kernel smut (RKS; T. horrida). We present the first genomes for four other species (T. bromi, T. fusca, T. goloskokovii, and T. rugispora), resulting in the largest and most diverse sample of Tilletia genomes studied to date. Depending on the species and strain, the assembly size ranged from 24.3 to 30.5 Mb and gene prediction resulted in 7138 to 8261 gene models per genome. Phylogenomic analyses with hundreds to thousands of genes revealed significant support for the relationships among certain Tilletia taxa and validated findings of previous molecular studies that employed a small number of genes. Further population-level analyses showed two distinct populations of DB and CB: T. controversa (DB) as a single population and another intermixed population of T. caries and T. laevis (CB). No evidence of geographic isolation was observed within these populations. Our phylogenomic analyses also supported previous multigene hypotheses that multiple lineages of Tilletia may cause RKS. Collectively, our results suggest that taxonomic revisions are needed for the RKS-causing pathogens and provide convincing evidence for formally recognizing the CB-causing taxa as one species, named T. caries (synonym T. laevis). Overall, our study significantly enhances genomic resources for Tilletia, offers insights into phylogenetic relationships and population structure, and provides whole genome sequences for future studies.

Genome-wide identification and biochemical characterization of glycoside hydrolase gene family members in Tilletia Horrida.

Rice kernel smut, caused by Tilletia horrida, is becoming an increasingly serious disease in hybrid rice planting, leading to production losses and quality decline of male-sterile rice varieties. Successful infection requires an efficient energy source that the pathogen obtains from rice plants, such as carbohydrates. Glycoside hydrolases (GHs), one of the largest sub-families in the cell wall-degrading enzyme family, play a key role in the infection progress of pathogens. To investigate their roles in facilitating infection, in this study, we identified and characterized genes encoding GH family proteins of T. horrida and further explored the functions and structures of these genes. Through genome-wide sequencing and bioinformatics analyses, 52 GH genes were identified from T. horrida, named ThGhd_1 to ThGhd_52. The subcellular location, conserved motifs, and structures of ThGhds were identified by bioinformatics analyses. Phylogenetic analysis revealed that ThGhds with similar domains clustered together, although some proteins clustered in different branches, which might reflect functional diversity. Protein-protein interaction network analysis revealed that ThGhds interact with partner proteins involved in reactive oxygen species signaling, protein kinase activity, and plant hormone signal transduction pathways. RNA-sequencing analysis showed that the expression of ThGhd genes responded differently at different infection time points, with dynamic changes detected during the T. horrida infection process, indicating that these genes are involved in interactions with rice and have potential roles in pathogenic mechanisms. The results of this study provide valuable resources for the structure elucidation of GH family proteins of T. horrida and can help to further elucidate their roles in pathogenesis.

Tilletia horrida glycoside hydrolase family 128 protein, designated ThGhd_7, modulates plant immunity by blocking reactive oxygen species production.

Tilletia horrida is an important soilborne fungal pathogen that causes rice kernel smut worldwide. We found a glycoside hydrolase family 128 protein, designated ThGhd_7, caused cell death in Nicotiana benthamiana leaves. The predicted signal peptide (SP) of ThGhd_7 targets it for secretion. However, loss of the SP did not affect its ability to induce cell death. The 23-201 amino acid sequence of ThGhd_7 was sufficient to trigger cell death in N. benthamiana. ThGhd_7 expression was induced and upregulated during T. horrida infection. ThGhd_7 localised to both the cytoplasm and nucleus of plant cells, and nuclear localisation was required to induce cell death. The ability of ThGhd_7 to trigger cell death in N. benthamiana depends on RAR1 (required for Mla12 resistance), SGT1 (suppressor of G2 allele of Skp1), and BAK1/SERK3 (somatic embryogenesis receptor-like kinase 3). Heterologous overexpression of ThGhd_7 in rice reduced reactive oxygen species (ROS) production and enhanced susceptibility to T. horrida. Further research revealed that ThGhd_7 interacted with and destabilised OsSGT1, which is required for ROS production and is a positive regulator of rice resistance to T. horrida. Taken together, these findings suggest that T. horrida employs ThGhd_7 to disrupt ROS production and thereby promote infection.

Functional Analyses of a Small Secreted Cysteine-Rich Protein ThSCSP_14 in Tilletia horrida

Tilletia horrida is a biotrophic basidiomycete fungus that causes rice kernel smut, one of the most significant diseases in hybrid rice-growing areas worldwide. Little is known about the pathogenic mechanisms and functions of effectors in T. horrida. Here, we performed functional studies of the effectors in T. horrida and found that, of six putative effectors tested, only ThSCSP_14 caused the cell death phenotype in epidermal cells of Nicotiana benthamiana leaves. ThSCSP_14 was upregulated early on during the infection process, and the encoded protein was secreted. The predicted signal peptide (SP) of ThSCSP_14 was required for its ability to induce the necrosis phenotype. Furthermore, the ability of ThSCSP_14 to trigger cell death in N. benthamiana depended on suppressing the G2 allele of Skp1 (SGT1), required for Mla12 resistance (RAR1), heat-shock protein 90 (HSP90), and somatic embryogenesis receptor-like kinase (SERK3). It is important to note that ThSCSP_14 induced a plant defense response in N. benthamiana leaves. Hence, these results demonstrate that ThSCSP_14 is a possible effector that plays an essential role in T. horrida-host interactions.

ThSCSP_12: Novel Effector in Tilletia horrida That Induces Cell Death and Defense Responses in Non-Host Plants

The basidiomycete fungus Tilletia horrida causes rice kernel smut (RKS), a crucial disease afflicting hybrid-rice-growing areas worldwide, which results in significant economic losses. However, few studies have investigated the pathogenic mechanisms and functions of effectors in T. horrida. In this study, we found that the candidate effector ThSCSP_12 caused cell necrosis in the leaves of Nicotiana benthamiana. The predicted signal peptide (SP) of this protein has a secreting function, which is required for ThSCSP_12 to induce cell death. The 1- 189 amino acid (aa) sequences of ThSCSP_12 are sufficient to confer it the ability to trigger cell death in N. benthamiana. The expression of ThSCSP_12 was induced and up-regulated during T. horrida infection. In addition, we also found that ThSCSP_12 localized in both the cytoplasm and nucleus of plant cells and that nuclear localization of this protein is required to induce cell death. Furthermore, the ability of ThSCSP_12 to trigger cell death in N. benthamiana depends on the (RAR1) protein required for Mla12 resistance but not on the suppressor of the G2 allele of Skp1 (SGT1), heat shock protein 90 (HSP90), or somatic embryogenesis receptor-like kinase (SERK3). Crucially, however, ThSCSP_12 induced a defense response in N. benthamiana leaves; yet, the expression of multiple defense-related genes was suppressed in response to heterologous expression in host plants. To sum up, these results strongly suggest that ThSCSP_12 operates as an effector in T. horrida-host interactions.

Multi-Locus Sequence Analysis Reveals Diversity of the Rice Kernel Smut Populations in the United States.

Rice (Oryza sativa) is the second leading cereal crop in the world and is one of the most important field crops in the US, valued at approximately $2.5 billion. Kernel smut (Tilletia horrida Tak.), once considered as a minor disease, is now an emerging economically important disease in the US. In this study, we used multi-locus sequence analysis to investigate the genetic diversity of 63 isolates of T. horrida collected from various rice-growing areas across in the US. Three different phylogeny analyses (maximum likelihood, neighbor-joining, and minimum evolution) were conducted based on the gene sequence sets, consisting of all four genes concatenated together, two rRNA regions concatenated together, and only ITS region sequences. The results of multi-gene analyses revealed the presence of four clades in the US populations, with 59% of the isolates clustering together. The populations collected from Mississippi and Louisiana were found to be the most diverse, whereas the populations from Arkansas and California were the least diverse. Similarly, ITS region-based analysis revealed that there were three clades in the T. horrida populations, with a majority (76%) of the isolates clustering together along with the 22 Tilletia spp. from eight different countries (Australia, China, India, Korea, Pakistan, Taiwan, The US, and Vietnam) that were grouped together. Two of the three clades in the ITS region-based phylogeny consisted of the isolates reported from multiple countries, suggesting potential multiple entries of T. horrida into the US. This is the first multi-locus analysis of T. horrida populations. The results will help develop effective management strategies, especially breeding for resistant cultivars, for the control of kernel smut in rice.

Comparative transcriptome analysis of Tilletia horrida infection in resistant and susceptible rice (Oryza sativa L.) male sterile lines reveals potential candidate genes and resistance mechanisms

Rice kernel smut (RKS), caused by the basidiomycete fungus Tilletia horrida, is one of the most devastating diseases affecting the production of male sterile lines of rice (Oryza sativa) worldwide. However, the molecular mechanisms of resistance to T. horrida have not yet been explored. In the present study, RNA sequencing analysis of rice male sterile lines, that are resistant and susceptible to RKS (Jiangcheng 3A and 9311A, respectively) was conducted after T. horrida infection. Transcriptomic analysis showed that a greater number of differentially expressed gene (DEGs) was observed in Jiangcheng 3A compared with 9311A after T. horrida inoculation. Furthermore, 4, 425 DEGs were uniquely detected in Jiangcheng 3A, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of these DEGs revealed that oxidoreductase activity, peroxidase activity, cutin, suberine and wax biosynthesis, and flavonoid biosynthesis were key pathways for T. horrida resistance. In summary and based on transcriptome analysis, we suggest a preliminary regulatory mechanism for Jiangcheng 3A cultivar resistance response to T. horrida inoculation.

Transcriptome analysis and whole genome re-sequencing provide insights on rice kernel smut (Tilletia horrida) pathogenicity

Rice kernel smut is an important disease caused by Tilletia horrida that affects the production of rice male sterile lines in most hybrid rice growing regions of the world. In this study, the pathogenicity of seven different strains of T. horrida isolated from different parts of China is described. Whole-genome re-sequencing of six T. horrida strains and transcriptome analysis of the reference genome T. horrida strain JY-521 were conducted at different times post inoculation (8, 12, 24, 48, and 72 h) early in the infection. The highest number of differentially expressed genes (DEGs) occurred at 8 h post inoculation. Based on Kyoto Encyclopedia of Genes and Genomes pathway analysis of the DEGs, autophagy processes and lipid degradation were key pathways for T. horrida pathogenicity. In three weakly pathogenic strains, CN-4, XJ-3, and SN-2, the single nucleotide polymorphisms and expression patterns of pathogenicity genes, including carbohydrate-active enzyme genes, pathogen-host interaction genes, effector genes, secondary metabolism-related genes, cytochrome P450 genes, ATP-binding cassette superfamily transporter genes, and G protein-coupled receptor genes, were also analyzed at different times during the infection. Thus, a new understanding of T. horrida pathogenicity was gained by exploring the potential mechanisms for weakened virulence in different strains of rice kernel smut, and a new foundation was provide for further studies on the infection mechanism and future control of this important rice disease.

Comparative secretome analysis of different smut fungi and identification of plant cell death-inducing secreted proteins from Tilletia horrida

BackgroundTilletia horrida is a basidiomycete fungus that causes rice kernel smut, one of the most important rice diseases in hybrid rice growing areas worldwide. However, little is known about its mechanisms of pathogenicity. We previously reported the genome of T. horrida, and 597 genes that encoded secreted proteins were annotated. Among these were some important effector genes related to pathogenicity.ResultsA secretome analysis suggested that five Tilletia fungi shared more gene families than were found in other smuts, and there was high conservation between them. Furthermore, we screened 597 secreted proteins from the T. horrida genome, some of which induced expression in host-pathogen interaction processes. Through transient expression, we demonstrated that two putative effectors could induce necrosis phenotypes in Nicotiana benthamiana. These two encoded genes were up-regulated during early infection, and the encoded proteins were confirmed to be secreted using a yeast secretion system. For the putative effector gene smut_5844, a signal peptide was required to induce non-host cell death, whereas ribonuclease catalytic active sites were required for smut_2965. Moreover, both putative effectors could induce an immune response in N. benthamiana leaves. Interestingly, one of the identified potential host interactors of smut_5844 was laccase-10 protein (OsLAC10), which has been predicted to be involved in plant lignification and iron metabolism.ConclusionsOverall, this study identified two secreted proteins in T. horrida that induce cell death or are involved in defense machinery in non-host plants. This research provides a useful foundation for understanding the interaction between rice and T. horrida.

Comparison of gene co-networks analysis provide a systems view of rice (Oryza sativa L.) response to Tilletia horrida infection.

The biotrophic soil-borne fungus Tilletia horrida causes rice kernel smut, an important disease affecting the production of rice male sterile lines in most hybrid rice growing regions of the world. There are no successful ways of controlling this disease and there has been little study of mechanisms of resistance to T. horrida. Based on transcriptional data of different infection time points, we found 23, 782 and 23, 718 differentially expressed genes (fragments per kilobase of transcript sequence per million, FPKM >1) in Jiangcheng 3A (resistant to T. horrida) and 9311A (susceptible to T. horrida), respectively. In order to illuminate the differential responses of the two rice male sterile lines to T. horrida, we identified gene co-expression modules using the method of weighted gene co-expression network analysis (WGCNA) and compared the different biological functions of gene co-expression networks in key modules at different infection time points. The results indicated that gene co-expression networks in the two rice genotypes were different and that genes contained in some modules of the two groups may play important roles in resistance to T. horrida, such as DTH8 and OsHop/Sti1a. Furthermore, these results provide a global view of the responses of two different phenotypes to T. horrida, and assist our understanding of the regulation of expression changes after T. horrida infection.

The pathogenic mechanisms of Tilletia horrida as revealed by comparative and functional genomics

Tilletia horrida is a soil-borne, mononucleate basidiomycete fungus with a biotrophic lifestyle that causes rice kernel smut, a disease that is distributed throughout hybrid rice growing areas worldwide. Here we report on the high-quality genome sequence of T. horrida; it is composed of 23.2 Mb that encode 7,729 predicted genes and 6,973 genes supported by RNA-seq. The genome contains few repetitive elements that account for 8.45% of the total. Evolutionarily, T. horrida lies close to the Ustilago fungi, suggesting grass species as potential hosts, but co-linearity was not observed between T. horrida and the barley smut Ustilago hordei. Genes and functions relevant to pathogenicity were presumed. T. horrida possesses a smaller set of carbohydrate-active enzymes and secondary metabolites, which probably reflect the specific characteristics of its infection and biotrophic lifestyle. Genes that encode secreted proteins and enzymes of secondary metabolism, and genes that are represented in the pathogen-host interaction gene database genes, are highly expressed during early infection; this is consistent with their potential roles in pathogenicity. Furthermore, among the 131 candidate pathogen effectors identified according to their expression patterns and functionality, we validated two that trigger leaf cell death in Nicotiana benthamiana. In summary, we have revealed new molecular mechanisms involved in the evolution, biotrophy, and pathogenesis of T. horrida.

Development and validation of microsatellite markers for Karnal bunt (Tilletia indica) and loose smut (Ustilago segetum tritici) of wheat from related fungal species

AbstractSimple sequence repeats (SSRs) are preferred molecular markers because of their abundance, robustness, high reproducibility, high efficiency in detecting variation and suitability for high‐throughput analysis. In this study, an attempt was made to mine and analyse the SSRs from the genomes of two seed‐borne fungal pathogens, viz Ustilago maydis, which causes common smut of maize, and Tilletia horrida, the cause of rice kernel smut. After elimination of redundant sequences, 2,703 SSR loci of U. maydis were identified. Of the remaining SSRS, 44.5% accounted for di‐nucleotide repeats followed by 29.8% and 2.7% tri‐ and tetranucleotide repeats, respectively. Similarly, 2,638 SSR loci were identified in T. horrida, of which 20.2% were di‐nucleotide, 50.4% tri‐ and 20.5% tetra‐nucleotide repeats. A set of 65 SSRs designed from each fungus were validated, which yielded 23 polymorphic SSRs from Ustilago and 21 from Tilletia. These polymorphic SSR loci were also successfully cross‐amplified with the Ustilago segetum tritici and Tilletia indica. Principal coordinate analysis of SSR data clustered isolates according to their respective species. These newly developed and validated microsatellite markers may have immediate applications for detection of genetic variability and in population studies of bunt and smut of wheat and other related host plants. Moreover, this is first comprehensive report on molecular markers suitable for variability studies in wheat seed‐borne pathogens.

Influence of Mineral and Organic Fertilizers on Rice Kernel Smut Disease

Influence of Mineral and Organic Fertilizers on Rice Kernel Smut Disease

Simple and rapid detection of Tilletia horrida causing rice kernel smut in rice seeds.

A simple and rapid method for the detection of Tilletia horrida, the causal agent of rice kernel smut, in rice seeds is developed based on specific polymerase chain reaction (PCR). To design the specific primers for the detection of T. horrida, partial sequences of internal transcribed spacer (ITS) DNA region of T. horrida, T. controversa, T. walkeri, T. ehrhartae, T. indica and T. caries were analyzed and compared. A 503-bp fragment was amplified with the designed primers from the T. horrida genomic DNA. However, no PCR product was obtained from the DNA of other five Tilletia species and 22 fungal plant pathogens tested in the present work indicating the specificity of the primers for the detection of T. horrida. The PCR was performed by directly using the spores, isolated from the 21 different rice seed samples, as template DNA. The T. horrida was detected in 6 of the samples, indicating that 28.6% of the rice samples were contaminated with the kernel smut pathogen. This simple PCR based diagnostic assay can be applied for the direct and rapid detection and identification of T. horrida to screen large numbers of rice seed samples.

Integrated control of rice kernel smut disease using plant extracts and salicylic acid

The effects of salicylic acid (SA) at three concentrations i.e. 2.5, 5 and 7 mM and plant extracts from pick tooth (Ammi visnaga), liquorice (Glycyrrhiza glabra), artemisia (Artemisia judaica), mint (Mentha viridis), clove (Syzygium aromaticum) and blue gum (Eucalyptus globulus) on the infection of rice kernel smut disease caused by Tilletia barclayana were studied. Spraying of rice plants with different concentrations of SA at seven days before infection was the most effective treatment against pathogen infection. Among all plant extract treatments, M. viridis and S. aromaticum were the most effective treatments. Additionally, our results showed increased levels of peroxidase, polyphenol oxidase, phenylalanine ammonia lyase and chitinase as well as total protein contents in the treated plants compared with the control. In conclusion, accumulations of these oxidative enzymes in plants treated with SA and plant extracts provide their role in the activation of induced resistance against T. barclayana.

English

The study was conducted to determine the effects of cultivars susceptibility to infection with the pathogenic isolates on the pathogenicity of rice kernel smut fungus (Tilletia barclayana). Rice (Oryza sativa L.) cultivars vary in susceptibility to kernel smut disease. The susceptibility of 18 commercial rice cultivars to T. barclayana was evaluated. Moreover, pathogenicity of 46 isolates of T. barclayana which had been isolated from six governorates in Egypt was studied using Giza 178 rice cultivar and Hybrid 1. The activities of certain oxidative enzymes, e.g. peroxidase (POX) and polyphenoloxidase (PPO) and total protein were determined in healthy and inoculated cultivars. Intron-exon splice junctions (ISJ) protocol was used to study the relationship among rice cultivars differing in their susceptibility to infection with the pathogen. Results showed that Giza 178 rice cultivar and Hybrid 1 were the most susceptible cultivars for rice kernel smut disease. Isolate no. 35 from the pathogenic fungus was the most aggressive isolate. Additionally, an increment in the defense-related enzymes and total protein was observed in rice cultivars as a result of inoculation with T. barclayana. ISJ technique showed the relationship between the studied rice cultivars and lines upon susceptibility to T. barclayana. It can also differentiate between the isolated T. barclayana isolates upon virulence on the studied rice cultivars and lines. Key words: Rice kernel smut disease, intron-exon splice junctions, rice, enzymes activity.

Tilletia barclayana: biologia e manejo em arroz irrigado

A cárie-do-arroz (Tilletia barclayana), doença considerada secundária na cultura do arroz irrigado, vem ganhando importância nos últimos anos devido aos níveis elevados de infestação e à redução do rendimento. A doença é favorecida por temperatura e umidade do ar elevadas, doses altas de nitrogênio e semeadura tardia. Para o manejo, recomenda-se uso de resistência varietal, semeadura na época recomendada e adubação equilibrada.

Enhancing disease resistances of Super Hybrid Rice with four antifungal genes

A plant expression vector harboring four antifungal genes was delivered into the embryogenic calli of '9311', an indica restorer line of Super Hybrid Rice, via modified biolistic particle bombardment. Southern blot analysis indicated that in the regenerated hygromycin-resistant plants, all the four antifungal genes, including RCH10, RAC22, beta-Glu and B-RIP, were integrated into the genome of '9311', co-transmitted altogether with the marker gene hpt in a Mendelian pattern. Some transgenic R1 and R2 progenies, with all transgenes displaying a normal expression level in the Northern blot analysis, showed high resistance to Magnaporthe grisea when tested in the typical blast nurseries located in Yanxi and Sanya respectively. Furthermore, transgenic F1 plants, resulting from a cross of R2 homozygous lines with high resistance to rice blast with the non-transgenic male sterile line Peiai 64S, showed not only high resistance to M. grisea but also enhanced resistance to rice false smut (a disease caused by Ustilaginoidea virens) and rice kernel smut (another disease caused by Tilletia barclayana).

Optimal application timing of simeconazole granules for control of rice kernel smut and false smut

We investigated the optimal timing of simeconazole (RS-2-(4-fluorophenyl)-1-(1H-1,2,4-triazol-1-yl)-3-trimethylsilylpropan-2-ol) application for controlling rice kernel smut in field trials in Miyagi Prefecture, Japan, using formulations of simeconazole (1.5% granules). The field tests revealed that a submerged application of simeconazole granules (450–600 g ai/ha) at 1–5 weeks before heading was highly effective against kernel smut, with treatments 1–2 weeks before heading being the most effective. Submerged application of the fungicide at 2–5 weeks before heading was also highly effective against false smut, with treatment 3 weeks before heading being the most effective. These periods overlap the timing for optimal application of simeconazole to control rice sheath blight and ear blight. Consequently, we concluded that treatment with simeconazole 2–3 weeks before heading can be a useful tool for controlling all four diseases.

Nonsystemic Bunt Fungi—Tilletia indica and T. horrida: A Review of History, Systematics, and Biology

The genus Tilletia is a group of smut fungi that infects grasses either systemically or locally. Basic differences exist between the systemically infecting species, such as the common and dwarf bunt fungi, and locally infecting species. Tilletia indica, which causes Karnal bunt of wheat, and Tilletia horrida, which causes rice kernel smut, are two examples of locally infecting species on economically important crops. However, even species on noncultivated hosts can become important when occurring as contaminants in export grain and seed shipments. In this review, we focus on T. indica and the morphologically similar but distantly related T. horrida, considering history, systematics, and biology. In addition, the controversial generic placement and evolutionary relationships of these two species are discussed in light of recent molecular studies.