- Significant differences in transcriptomes of the susceptible and resistant genotypes were observed with dispersion distance and hierarchical cluster tree analyses. - Of the total 33, 032 genes predicted in the sorghum genome, 19,593 were induced by C. - The resistant line was marked by significant reprogramming of the transcriptome at 24 h post inoculation (hpi), and a decrease at 48 hpi, whereas the susceptible line displayed continued changes in gene expression concordant with elevated fungal growth in the susceptible genotype. - Genes encoding immune receptors, MAPKs, pentatricopeptide repeat proteins, and WRKY transcription factors were induced in the resistant genotype. - The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. - Full list of author information is available at the end of the article. - Sorghum (Sorghum bicolor (L.) Moench) is one of the most important cereal crops cultivated mainly in warm tropical regions of the world as a major source of grain and feed for livestock [1, 2]. - Trotter) [7], is one of the most devas- tating diseases of the crop. - The fungus exhibits a hemibiotrophic mode of nutrition with early biotrophic phase followed by necro- trophic phase characteristics of other species in the genus. - Accordingly, resist- ance cultivars have been developed in the last decades and used for crop production [12]. - In all cases, neither the compo- nents of the host nor the pathogen derived elicitors are known. - Gene expression data and analyses of secondary metabolites implicate genes in the PAL and flavonoid pathways for C. - Changes in gene expression is one of the most rapid host responses early after infection and could suggest critical genetic players in resistance or susceptibility to infection. - A battery of defense responses are transcrip- tionally regulated resulting in the accumulation of PR proteins, secondary metabolites and signaling compo- nents. - In the current study, we analyzed genome wide mRNA and miRNA expression profiles of resistant and suscep- tible sorghum genotypes in response to C. - The distinct diseases responses of the two genotypes was confirmed by the extent of fungal sequences recovered in the susceptible but not in the resistant cultivar.. - Expression of diverse genes implicated in the biosynthesis of secondary metabolites, defense signaling pathways, and photosynthesis were altered in response to the fungus. - For each sample, more than 90% of the reads could be uniquely mapped to the sorghum reference genome. - Fur- ther, hierarchical clustering analysis of the sequence data across samples indicated that SC283 and TAM428 form distinct groups (Fig. - Sorghum genes differentially expressed in response to Colletotrichum sublineolum. - In the present study, genes with differential expression between susceptible and resistance plants (FDR <. - In total, 19,593 of the 33,032 sorghum genes showed altered expression in response to C. - 0.01) at least in one of the treatments (Fig. - The expression patterns observed in the RNA-seq and qRT-PCR assay were largely the same (Figure S1) which demonstrated the reliability of the data produced through RNA-seq in this study.. - Functional classifications of genes differentially expressed in response to Colletotrichum sublineolum. - In order to understand the possible biological function of genes differentially expressed in the two sorghum lines, the functional classifications of DEGs (15,512) were analyzed using the InterPro database. - 1 Disease response phenotypes and global evaluation of the RNA-seq data in sorghum lines SC283 and TAM428. - In the current study, 252 RLKs with LRR domain (PF08263 and PF00560), 77 Lectin_legB (PF00139), 76 S-locus kinase with S_locus_. - A total of 125 NBs-LRR genes were differentially expressed in response to C. - The number of Table 1 Sequencing metrics of the 18 RNA-seq libraries. - DEGs at 24 hpi were comparable in the two genotypes, with 79 DEGs in SC283 and 73 in TAM428. - sublineolum in the susceptible genotype.. - Among these, chitinases are involved in the degradation of chitin, which is the primary structural com- ponent of fungal cell walls. - We identified a total of 95 WRKY genes in the sorghum reference genome version 3.1. - One of the sorghum WRKY70 genes (Sobic.001G381300), was up-regulated 8-fold at 24 hpi in TAM428. - 2 Overview of genes differentially expressed in response to Colletotrichum sublineolum in sorghum. - b Domain enrichment analysis of proteins encoded by genes differentially expressed in response to C. - In sorghum, a total of 119 MAPK pathway genes were identified in the reference genome version 3.1. - MAPK cascade genes were induced at 24 hpi but not differentially expressed at 48 hpi in SC283. - Differentially expressed MAPK genes were identified both at 24 hpi and 48 hpi in TAM428. - Expression of Pentatricopeptide repeat genes in response to Colletotrichum sublineolum. - We identified 265 PPRs in the sorghum reference genome version 3.1.. - sublineolum inoculation in SC283 and TAM428 (Table S6). - Regardless of the genotype and infection, the size distribution of small RNAs peaked at 22 and 24 nt. - Differential expression of sorghum miRNAs in response to Colletotrichum sublineolum. - In the current study, a total of 1122 miRNA target genes were identified in the sorghum genome based on the 69 previously known and 123 novel and unique candidate se- quences of sorghum miRNAs that meet our filtering criteria (Table S8). - The 62 potential miRNA target genes were differentially expressed in response to C.. - A gene ontology (GO) functional classification analysis to understand the functions of the identified targeted genes resulted in a total of 37 GO terms including 12 cellular components, 15 biological process, and 10 molecular functions (Fig. - 4 Expression profiling of miRNAs in response to Colletotrichum sublineolum. - a Hierarchical clustering of differentially expressed miRNAs. - The original expression values of the miRNAs were normalized using Z-score. - The fungus causes one of the most widespread diseases of sorghum. - The later phase of the diseases involves extensive growth of secondary hyphae and enhanced fungal colonization of the host in susceptible genotypes.. - Two contrasting sorghum genotypes, highly resistant and susceptible genotypes with distinct responses were used in the global expression profiling experiment described here. - Interestingly, significant reprogramming of tran- scripts continues in the susceptible TAM428 even at 48 hpi while in the resistant genotype, a rapid and signifi- cant drop was observed in the number of genes that are up or down regulated. - Rapid and extensive changes in gene expression were observed in the resistant line at 24. - This is consist- ent with continued fungal growth, and disease symptom observed in the susceptible but not in the resistant culti- var. - A rapid increase in expression of genes encoding MAPKs, pentatricopeptide repeat proteins (PPR), photo- synthesis and tetrapyrole biosynthesis genes were observed in the resistant which were repressed in the susceptible line.. - Broadly, sorghum genes differentially expressed in response to C. - Interestingly, in the resistant line, there was a greater proportion of DEGs that encode proteins involved tetrapyrrole biosynthesis, photosynthesis, cell wall, and defense signaling. - Interestingly, DEGs that correlated with resistance encode a range of proteins in- cluding pathogen recognition receptors, downstream com- ponents such as MAPKs, transcription factors, and genes involved in the synthesis of antimicrobial metabolites. - Caution should also be exercised in the interpretation of the data and explaining the phenotypes of the two lines to specific pathogen induced genes. - It is, however, clear that in the susceptible line, due to the elevated fungal growth many genes were induced especially at 48 hpi, some of which may mark a cellular perturbation rather than a specific immune response function. - Despite the widespread and damaging nature of diseases caused by species in the genus Colletotrichum the host response pathways are understudied. - Although both quantitative and race specific resistances have been de- scribed, neither the specific components of the pathogen nor the specific genes in the host have been determined.. - Some of the DEGs identified may contribute to resistance through modulation of HR, accumulation of reactive oxygen species, enhancement of cell wall, and biosynthesis of antimicrobial compounds. - The increased expression of genes encoding some of the classical PR proteins such as chitinases, in the susceptible genotype do not appear to explain the disease responses differences in the two lines.. - It is still possible that upregulation of sorghum chitinases may contribute to quantitative resistance in the suscep- tible background but are likely not sufficient to confer full resistance consistent with the disease responses and phenotypes of TAM428.. - Regardless of the particular path- way, plant immunity to fungal pathogens converge into common down-stream immune response such as oxida- tive burst, accumulation of phytoalexins and PR proteins. - In addition to various gene families and functional groups described in the preceding sections, a large num- ber of genes encoding Pentatricopeptide-repeat proteins (PPR) were identified to be regulated during fungal in- fection. - PPRs are a family of proteins commonly found in the plant kingdom that are distinguished by the pres- ence of tandem degenerate PPR motifs (35-amino acid sequence motif) and by the relative lack of introns in PPR encoding genes [57]. - Despite the large size of the protein family, genetic data suggest that there is little or no functional redundancy between PPR proteins in Arabidopsis. - In the recent release of miRBase (version 21), a total of 241 miRNAs have been identified based on the sorghum reference genome (BTX623, Sorbi1) [65]. - Previously, miRNAs and secondary siRNAs have been implicated in NBs-LRR gene regulation and pathogen resistance in the Solanaceae [70]. - In the same study, a number of differentially expressed miRNAs that are differentially regulated by the bacterial PAMP flg22 were identified. - Similarly, a rapid increase in expression of MAPKs, photosynthesis and tetrapyrole biosynthesis genes in the resistant genotype was observed. - the same strains, and detailed molecular and phenotype characterization of the two genotypes is described [74].. - To filter out weakly expressed genes, only those genes with a minimum expression level of 1 RPKM (reads per kilobase per million) in three replicates were included in the analysis. - Functional classification of differentially expressed genes To evaluate potential function of the large number of genes with differential expression in response to C.. - The mixture was incubated at 42 °C for 1 h, followed by inactivation of the reverse transcriptase at 90 °C for 10 min. - For data analysis, the mean Ct value of the target gene was normalized to the average Ct value of sorghum actin gene using the ΔCt method implemented in the CFX manager software (Bio-Rad Laboratories, USA).. - 10 tran- scripts per million, in at least one of the twelve libraries.. - Newly identified miRNAs were considered as candidate only when detected consistently across the three replicates of the libraries with similar treatment in SC283 or TAM428.. - WRKY genes differentially expressed in susceptible and resistant genotypes of sorghum. - MAPK genes differentially expressed in susceptible and resistant genotypes of sorghum. - Pentatricopeptide (PPR) genes differentially expressed in susceptible and resistant genotypes of sorghum. - List of all miRNA target genes identified in the sorghum genome. - Additional file 9: Table S9.Target genes identified, their classification and identification of the function of gene products with different databases. - List of differentially expressed miRNA target genes and their corresponding miRNAs in sorghum. - Validation of RNA-seq data through qRT- PCR analyses of selected differentially expressed genes in response to C.. - The filters are described in the main text and summarized in the flowchart on the left. - On the right are the re- sults of the application of the pipeline in sorghum. - DEGs: Differentially expressed genes. - The contents are the sole responsibility of the authors and do not necessarily reflect the views of USAID or the United States Government. - Ultrastructure of the infection of Sorghum bicolor by Colletotrichum sublineolum. - Functional annotation of the transcriptome of Sorghum bicolor in response to osmotic stress and abscisic acid. - Genome-wide analysis of the rice PPR gene family and their expression profiles under different stress treatments. - MicroRNA- mediated regulation of gene expression in the response of rice plants to fungal elicitors. - accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions
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