Transcriptome analysis of wheat spikes in response to Tilletia controversa Kühn which cause wheat dwarf bunt

Wheat dwarf bunt is caused by Tilletia controversa Kühn, which is one of the most destructive diseases of wheat worldwide. To explore the interaction of T. controversa and wheat, we analysed the transcriptome profile of spikes of the susceptible wheat cultivar Dongxuan 3, which was subjected to a T. controversa infection and a mock infection. The results obtained from a differential expression analysis of T. controversa-infected plants compared with mock-infected ones showed that 10,867 out of 21,354 genes were upregulated, while 10,487 genes were downregulated, and these genes were enriched in 205 different pathways. Our findings demonstrated that the genes associated with defence against diseases, such as PR-related genes, WRKY transcription factors and mitogen-activated protein kinase genes, were more highly expressed in response to T. controversa infection. Additionally, a number of genes related to physiological attributes were expressed during infection. Three pathways were differentiated based on the characteristics of gene ontology classification. KEGG enrichment analysis showed that twenty genes were expressed differentially during the infection of wheat with T. controversa. Notable changes were observed in the transcriptomes of wheat plants after infection. The results of this study may help to elucidate the mechanism governing the interactions between this pathogen and wheat plants and may facilitate the development of new methods to increase the resistance level of wheat against T. controversa, including the overexpression of defence-related genes.

Scientific Reports | (2020) 10:21567 | https://doi.org/10.1038/s41598-020-78628-0 www.nature.com/scientificreports/ (Illumina, San Diego, CA, USA); similarly, second-strand complementary DNA was obtained using RNase H and DNA polymerase. A QIAquick PCR kit was employed for purification of cDNA fragments. Then, these cDNA fragments were washed with EB buffer for the addition of end-repair poly (A) and ligated with special sequencing adapters. The final cDNA library was constructed by purification of the cDNA small fragments, which were enriched by PCR products.
Library examination and sequencing. The constructed cDNA library was validated by using the Qubit RNA Assay Kit in Qubit 3.0 for initial quantification. The insert size was determined using a Bioanalyzer 2100 Agilent system (Agilent, Santa Clara, CA, USA). Furthermore, the insert was amplified using qPCR (7500, ABI, USA). The clustering of every sample was performed on Generation systems (Illumina, USA) following a previously described protocol. The prepared library was loaded onto an Illumina HiSeq X Ten platform with 150-bp paired-end technology.
Quality control and mapping. The raw data of this experiment were further processed using Trimmomatic (trimmer for Illumina sequence data, Version 0.32) 55 . The reads containing adapter sequences and reads with low quality (those in which more than 50% of bases presented quality of ≤ 10) and poly-N (unrecognized bases) were removed to obtain clean reads. Every downstream analysis was performed based on clear data with significantly high quality. The clean reads were mapped to the reference genome (https ://www.ebi.ac.uk/ena/ data/view/GCA_90051 9105.1) using hisat2 56 with the parameters set by the system.

Gene-level quantification and identification of differentially expressed genes (DEGs). The
FPKM value of every gene was analysed and calculated by using cufflinks (version 2.2.1) 57,58 , and every read count of all genes was obtained by HTSeq-count 59 . Additionally, the DEGs of this study were recognized by using the DESeq 60 technique. Furthermore, the FDR ˂ 0.05, and at least a two-fold change (> 1 or < − 1 in log 2 ratio value) was set as the threshold for DEGs. Hierarchical cluster analysis (HCA) of all DEGs was performed to explore gene expression patterns.

Confirmation of T. controversa infection in wheat plants. After inoculation with T. controversa,
symptoms appeared in the spike with the formation of black teliospores. Specifically, the heads of infected plants were thicker and wider and squarrose. The florets were filled with bunt balls (sori). These bunt balls replaced the grains and produced black teliospores with a rotten fish-like odour. To identify the genes with changed expression following infection by T. controversa and to detect any change in gene expression levels after infection, RNA extraction was performed to investigate the changes in wheat at the transcriptomic level.  www.nature.com/scientificreports/ were differentially expressed. The differential expression of genes in CK and T. controversa indicates the genetic difference between mock-and T. controversa-infected plants (Fig. 1). Additionally, three biological replicates of every sample were clustered together. Sample-to-sample clustering analysis demonstrated that the gene expression level between replicates was reproducible and that batch effects were controlled (Fig. 2). Furthermore, principal component analysis (PCA) was performed for both mock-and T. controversa-infected samples. The mock was located around the junction of the second and third quadrants, and the T. controversa infection was located around the junction of the first and fourth quadrants, indicating that there is good reproducibility among the biological replicates of the same treatments but differences between the treatments (Fig. 3).

Identification of differentially expressed genes (DEGs).
The differentially expressed genes were recognized in T. controversa-infected and mock-infected libraries. In this comparison, 10,867 (up-regulated) and 10,487 (down-regulated) genes were expressed ( Fig. 4; Table S2). To elucidate the transcriptional changes occurring after T. controversa infection, we demonstrated the expression pattern by using hierarchical cluster-  Gene ontology (GO) enrichment analysis of DEGs. GO enrichment analysis of DEGs can demonstrate the function of genes. GO was categorized into three main domains based on their functions: biological process, cellular component and molecular function. Our results showed that in the biological process category, during the comparison of T. controversa-infected and mock-infected plants, GO was mainly associated with cellular process, metabolic process, multicellular organismal process, regulation of biological process, reproduction, reproduction process, response to stimulus, and single-organism process. Meanwhile, in the cellular component category, DEGs were primarily associated with cell, cell part, extracellular region, macromolecular complex, membrane, membrane part, organelle, and organelle part. In the molecular function category, DEGs primarily mapped with binding, catalytic activity, enzyme regulator activity, nucleic acid binding transcription factor activity, structural membrane activity and transporter activity (Fig. 6). In contrast, biological adhesion, cell killing and locomotion were observed to be uniquely enriched in biological process, while extracellular matrix, extracellular matrix part and nucleoid were uniquely enriched in cellular component and metallochaperone activity, protein tag, receptor regulator activity and translation regulator activity were determined to be uniquely enriched in molecular function (Fig. 6).
KEGG enrichment analysis of DEGs. KEGG analyses were performed to better understand the molecular associations among the DEGs. For DEGs between T. controversa-infected and mock-infected plants, 205 different pathways were identified (Table S3). However, the top 20 KEGG enrichment pathways of peroxisomes, FoxO signalling pathway, DNA replication, biosynthesis of amino acids, carbon metabolism, carbon fixation in photosynthetic organisms, methane metabolism, glyoxylate and dicarboxylate metabolism, chloroalkane and chloroalkene degradation, pyruvate metabolism, starch and sucrose metabolism, lysine degradation, valine, leucine and isoleucine degradation, glycine-serine-and threonine metabolism, photosynthesis-antenna proteins, cutin, suberine and wax biosynthesis, fatty acid degradation and glycolysis/gluconeogenesis were primarily activated. The pathway of biosynthesis of siderophore group nonribosomal peptides was activated slightly during the interaction (Fig. 7).  (Table S4).  (Table S5).

Quantitative real-time PCR.
To verify the changes in expression level exhibited by the identified DEGs in response to T. controversa infection, the expression levels of eight genes examined by quantitative real-time PCR (qRT-PCR). The expression pattern of validated genes was similar to the results obtained from RNA-Seq ( Table 2). The qRT-PCR results showed that seven genes were up-regulated and Lipase was determined to be down-regulated by both RNA-Seq and qRT-PCR analyses. Hence, the qRT-PCR results confirmed the RNA-Seq data.

Discussion
In the earliest studies of T. controversa and wheat, microscopic studies were performed to observe the structural changes in wheat after infection. In this research, we examined the plant defence responses in wheat following infection by T. controversa. We investigated interactions between susceptible wheat cultivars (Dongxuan 3) and T. controversa. We hypothesized that in these interactions, wheat transcriptomic changes are associated with the plant response to infection; thus, diseases appeared. Therefore, in this experiment, we employed RNA-Seq to perform a transcriptomic study of wheat following T. controversa infection and analysed the changes in the expression levels of genes in mock-and T. controversa-infected wheat plants. Our results demonstrated significantly differentially expressed genes between mock-and T. controversa-infected libraries.
Pathogenesis-related (PR) genes play a key role in the defence mechanisms of plants against biotic factors [64][65][66][67] . Overexpression of the PR genes encrypting pathogenesis-related proteins, thaumatin-like proteins, chitinases, peroxidases and glucanases increases resistance to various pathogens in different crops 11,68,69 . We compared the transcription level between pathogen-infected and mock-infected plants at the flowering stage. The transcription levels of 215 PR genes were changed by T. controversa infection (Table S4), including seven pathogenesis-related proteins, twenty-three thaumatin-like proteins, twenty-eight chitinase proteins, one hundred twenty-one peroxidase proteins and thirty-six glucanase proteins. Most of the one hundred thirty-five genes were up-regulated, and eighty were down-regulated. Together, these defence-related proteins might play a role in disease suppression against T. controversa.
Pattern recognition receptors (PRRs) have been developed to recognize MAMPs/PAMPs (microbe/pathogenassociated molecular patterns), which are conserved small molecules present across a broad classification of microbes. These plant receptors belong to the receptor-like kinase (RLK) family [79][80][81] . In our study, 761 different protein kinases were expressed in wheat ears after T. controversa infection (Table S6). Most kinase proteins were down-regulated after T. controversa infection. Interestingly, cysteine-rich receptor-like protein kinase, leucinerich repeat receptor protein kinase, probable inactive leucine-rich repeat receptor, probable LRR receptor-like www.nature.com/scientificreports/ serine/threonine-protein kinase, probable receptor-like protein kinase and proline-rich receptor-like protein kinase were almost completely down-regulated after infection. These consequences indicate that T. controversa releases and carries effectors into wheat ear cells during attack to overcome the immune signalling pattern of plants, thereby leading to DBW. Our results concerning kinase proteins were similar to those obtained by Hosseini 82 in their studies of another pathogen. Mitogen-activated protein kinase (MAPK) genes have been investigated in the plant response to fungal pathogens 83 . In our study, we found that fifty MAPK genes were expressed after T. controversa infection (Table S6), which suggests that MAPK genes play a role in wheat resistance to T. controversa infection. www.nature.com/scientificreports/ According to GO enrichment analysis of differentially expressed genes in mock-infected and T. controversainfected libraries, GO was categorized into three main domains based on their functions: biological process, cellular component, and molecular function. (Fig. 6). The results showed that cellular process and metabolic process had the highest number of DEGs during plant pathogen interactions. The GO results support the hypothesis that plant pathogens cause changes in the primary (plant growth and development) and secondary (induction of defence programme) metabolisms of the plants. Once plants are infected by pathogens, plant metabolism tends to expend more energy on plant defence activation compared with growth, development, cellular maintenance and reproduction [84][85][86][87] . This phenomenon suggests that the DEG metabolic process is related to the pathogenic mechanism of T. controversa, as well as plant-pathogen interactions.
KEGG pathway analysis demonstrated that most DEGs were characterized by carbon metabolism, starch and sucrose metabolism, biosynthesis of amino acids and glycolysis/gluconeogenesis pathways ( Fig. 7; Table S3). Biosynthesis of the siderophore group nonribosomal peptide pathway included down-regulated genes that encode ferric iron acquisition in many microorganisms. Iron plays a role in microbial proliferation and growth. Thus, iron are supposed to play a key role in disease development 88,89 . Microbial ferric iron reductase is a key enzyme that degrades ferric iron in microbes 90 . Our results are in keeping with the down-regulation of microbial ferric iron reductase due to fungal infection 91,92 . The results obtained from KEGG analysis of DEGs showed that significant DEGs were annotated to 205 different pathways, which suggests that T. controversa infection affects various biological functions of wheat (Table S3). Dwarfing is an important symptom of DBW; changes in gene expression related to morpho-physiological characteristics, especially plant height, are notable in wheat infected by T. controversa and might be involved in dwarfing symptoms. Our results showed that the expression of cytochrome P450 changed due to T. controversa infection (Table S3). Cytochrome P450 has various biosynthetic activities and plays a positive role in plant growth and development by producing gibberellins and brassinosteroids 93,94 .
Overall, our findings provide a genome-wide gene expression profile for wheat plants infected with T. controversa and may help to elucidate the regulatory mechanisms governing the response of wheat to this pathogen.