RNA-seq analysis of resistant and susceptible sub-tropical maize lines reveals a role for kauralexins in resistance to grey leaf spot disease, caused by Cercospora zeina

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dc.contributor.author Meyer, Jacqueline
dc.contributor.author Berger, David Kenneth
dc.contributor.author Christensen, Shawn A.
dc.contributor.author Murray, Shane L.
dc.date.accessioned 2018-04-10T08:04:39Z
dc.date.available 2018-04-10T08:04:39Z
dc.date.issued 2017-11
dc.description Additional file 1: Grey leaf spot (GLS) severity quantitative trait loci (QTLs) identified for the CML444 X SC Malawi maize recombinant inbred line population identified in the Baynesfield trial. List of disease severity QTL identified in RIL population [21, 50] and corresponding alleles in RIL165 and RIL387 en_ZA
dc.description Additional file 2: RNA-Seq data analysis pipeline. Read quality was evaluated by the fastQC application v0.11.2. Illumina 1.5 encoded quality scores (Q) were converted to Sanger scale (phred) using FASTQ Groomer Galaxy v1.0.4. Thereafter, sequence reads were mapped to v2 of the B73 reference genome (5b.60 annotation; sequence obtained from Phytozome v9.1), using TopHat v2.0.9 (http://ccb.jhu.edu/software/tophat/ index.shtml/), by implementing Bowtie2 v1.0.0. Cufflinks v2.0.2 (http:// cole-trapnell-lab.github.io/cufflinks//) was used to calculate transcript abundance, reported as fragments per kilobase pair of exon model per million fragments mapped (FPKM). Transcript assemblies were merged with the reference annotation into a single .gtf file using Cuffmerge. Differential expression analysis was conducted on the merged file using Cuffdiff with a False Discovery Rate (FDR) threshold set to 0.05 en_ZA
dc.description Additional file 3: Expression levels, protein annotation and GO annotation of differentially expressed genes. Statistically significant differentially expressed genes between the two genotypes are reported. The Log2FC derived from the comparisons of the fragments per kilobase of transcript per million fragments mapped (FPKM) expression values of RIL165 vs RIL387 is depicted. For each gene, the putative annotation of the protein according to the RefSeq database (provided by the National Center for Biotechnology Information (NCBI), www.ncbi.nlm.nih.gov/ refseq/), Uniprot (Universal Protein Resource; www.uniprot.org) and GenBank sequence database (provided by the NCBI, www.ncbi.nlm.nih.gov/genbank/) is described. Gene ontology terms mapped to each gene by AgriGo are included, in addition to the chromosomal positions of the genes in v2 of the B73 reference genome. Where the DE gene overlapped with the genomic position of a disease severity QTL [21, 50] this was indicated en_ZA
dc.description Additional file 4: Comparison of RNA-Seq and RT-qPCR expression analyses of genes between RIL165 and RIL387. Expression profiles of (a) entcopalyl diphosphate synthase 2 (GRMZM2G044481), (b) syn-copalyl diphosphate synthase (GRMZM2G068808), (c) Terpene synthase 6 (GRMZM2G127087_T03), (d) β-glucosidase1 (GRMZM2G031660), (e) Bx3 (GRMZM2G167549), (f) Bx5 (GRMZM2G063756), (g) Bx8 (GRMZM2G085054), and (h) Bx9 (GRMZM2G161335) is depicted en_ZA
dc.description Additional file 5: Significant enriched GO terms and associated genes responsive to C. zeina infection in a susceptible (RIL165) maize line. Gene ontology enrichment analysis was carried out using agriGO v1.2 of statistically significant differentially expressed genes with a Log2FC > 1 or < −1. Singular enrichment analysis (SEA) was performed using a hypergeometric test, Hochberg FDR adjustment method parameters, a significance level of 0.05, and a minimum number of five mapped entries using the complete set of gene ontology terms en_ZA
dc.description Additional file 6: Overview of pathways where differentially expressed genes participate as reported by MADIBA. Up-regulated gene products were mapped onto metabolic pathways using the KEGG representation. The number of enzymes in each pathway is portrayed for both RIL165 and RIL387 en_ZA
dc.description Additional file 7: Photographs depicting GLS disease progression in RIL165 and RIL387 greenhouse material inoculated with C. zeina. Material was harvested at three time points based on development of GLS disease symptoms: immediately after inoculation (0dpi, control), development of chlorotic spots (14 dpi) and development of grey leaf spot lesions (24 dpi for RIL165 and 28 dpi for RIL387) en_ZA
dc.description Additional file 8: Zealexin defences are induced in response to C. zeina. Leaves were treated with a spore solution (3 × 105 conidia/ml) and harvested at 0 days post inoculation (dpi), 14dpi and 24 or 28dpi (RIL165 and RIL387 respectively). The metabolite content of each sample was analysed using gas chromatography/chemical ionization – mass spectrometry. Zealexins were quantified based on the internal standard 13C18-linolenic acid and presented in ng/μg FW. Average levels of total zealexin metabolites depicted for RIL165 and RIL387 (n = 3–5; ±SEM) en_ZA
dc.description Additional file 9: Biosynthesis of benzoxazinoids in maize. The biosynthetic pathway of DIMBOA is depicted as per [112]. The expression profiles of DIMBOA biosynthetic genes in glass house leaf material is presented en_ZA
dc.description Additional file 10: Primer sequences and descriptive information of genes studied en_ZA
dc.description.abstract BACKGROUND : Cercospora zeina is a foliar pathogen responsible for maize grey leaf spot in southern Africa that negatively impacts maize production. Plants use a variety of chemical and structural mechanisms to defend themselves against invading pathogens such as C. zeina, including the production of secondary metabolites with antimicrobial properties. In maize, a variety of biotic and abiotic stressors induce the accumulation of the terpenoid phytoalexins, zealexins and kauralexins. RESULTS: C. zeina-susceptible line displayed pervasive rectangular grey leaf spot lesions, running parallel with the leaf veins in contrast to C. zeina-resistant line that had restricted disease symptoms. Analysis of the transcriptome of both lines indicated that genes involved in primary and secondary metabolism were up-regualted, and although different pathways were prioritized in each line, production of terpenoid compounds were common to both. Targeted phytoalexin analysis revealed that C. zeina-inoculated leaves accumulated zealexins and kauralexins. The resistant line shows a propensity toward accumulation of the kauralexin B series metabolites in response to infection, which contrasts with the susceptible line that preferentially accumulates the kauralexin A series. Kauralexin accumulation was correlated to expression of the kauralexin biosynthetic gene, ZmAn2 and a candidate biosynthetic gene, ZmKSL2. We report the expression of a putative copalyl diphosphate synthase gene that is induced by C. zeina in the resistant line exclusively. DISCUSSION : This study shows that zealexins and kauralexins, and expression of their biosynthetic genes, are induced by C. zeina in both resistant and susceptible germplasm adapted to the southern African climate. The data presented here indicates that different forms of kauralexins accumulate in the resistant and susceptible maize lines in response to C. zeina, with the accumulation of kauralexin B compounds in a resistant maize line and kauralexin A compounds accumulating in the susceptible line. en_ZA
dc.description.department Plant Production and Soil Science en_ZA
dc.description.librarian am2018 en_ZA
dc.description.sponsorship The Technology Innovation Agency (TIA), National Research Foundation (NRF) and the Genomics Research Institute at the University of Pretoria (UP), South Africa. en_ZA
dc.description.uri https://bmcplantbiol.biomedcentral.com en_ZA
dc.identifier.citation . & Murray, S.L.. et al. 2017, 'RNA-seq analysis of resistant and susceptible sub-tropical maize lines reveals a role for kauralexins in resistance to grey leaf spot disease, caused by Cercospora zeina', BMC Plant Biology, vol. 17, art. no. 197, pp. 1-20. en_ZA
dc.identifier.issn 1471-2229 (online)
dc.identifier.other 10.1186/s12870-017-1137-9
dc.identifier.uri http://hdl.handle.net/2263/64447
dc.language.iso en en_ZA
dc.publisher BioMed Central en_ZA
dc.rights © The Author(s). 2017. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/). en_ZA
dc.subject Grey leaf spot en_ZA
dc.subject Cercospora zeina en_ZA
dc.subject Zea mays en_ZA
dc.subject Phytoalexin en_ZA
dc.subject Kauralexin en_ZA
dc.subject RNA-Seq en_ZA
dc.title RNA-seq analysis of resistant and susceptible sub-tropical maize lines reveals a role for kauralexins in resistance to grey leaf spot disease, caused by Cercospora zeina en_ZA
dc.type Article en_ZA


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