Simultaneous Transcriptome Analysis of Sorghum and Bipolaris sorghicola by Using RNA-seq in Combination with De Novo Transcriptome Assembly

Abstract

The recent development of RNA sequencing (RNA-seq) technology has enabled us to analyze the transcriptomes of plants and their pathogens simultaneously. However, RNA-seq often relies on aligning reads to a reference genome and is thus unsuitable for analyzing most plant pathogens, as their genomes have not been fully sequenced. Here, we analyzed the transcriptomes of Sorghum bicolor (L.) Moench and its pathogen Bipolaris sorghicola simultaneously by using RNA-seq in combination with de novo transcriptome assembly. We sequenced the mixed transcriptome of the disease-resistant sorghum cultivar SIL-05 and B. sorghicola in infected leaves in the early stages of infection (12 and 24 h post-inoculation) by using Illumina mRNA-Seq technology. Sorghum gene expression was quantified by aligning reads to the sorghum reference genome. For B. sorghicola, reads that could not be aligned to the sorghum reference genome were subjected to de novo transcriptome assembly. We identified genes of B. sorghicola for growth of this fungus in sorghum, as well as genes in sorghum for the defense response. The genes of B. sorghicola included those encoding Woronin body major protein, LysM domain-containing intracellular hyphae protein, transcriptional factors CpcA and HacA, and plant cell-wall degrading enzymes. The sorghum genes included those encoding two receptors of the simple eLRR domain protein family, transcription factors that are putative orthologs of OsWRKY45 and OsWRKY28 in rice, and a class III peroxidase that is a homolog involved in disease resistance in the Poaceae. These defense-related genes were particularly strongly induced among paralogs annotated in the sorghum genome. Thus, in the absence of genome sequences for the pathogen, simultaneous transcriptome analysis of plant and pathogen by using de novo assembly was useful for identifying putative key genes in the plant–pathogen interaction.

Dual RNA-seq of pathogen and host

“…Recently several studies have reported the application of joint RNA-seq to infection models Petre et al, 2012;Tierney et al, 2012;Yazawa et al, 2013), however, due to the fundamental differences between bacterial and eukaryotic transcriptomes such analyses have been restricted to hosts infected with eukaryotic parasites. The ubiquitous polyadenylation of eukaryotic mRNAs and the sequence similarity of ribosomal transcripts facilitates the experimental protocol as it enables the poly(A) enrichment or ribosomal depletion for host and pathogen in a single step.…”

A highly multiplexed and sensitive RNA-seq protocol for simultaneous analysis of host and pathogen transcriptomes

“…RNAseq analysis of host-pathogen interactions has already been successfully applied for eukaryotic pathogens [11][12][13][14][15] . Transcripts of eukaryotic pathogens, unlike bacteria, are polyadenylated, and occur with comparable abundance to the hosts.…”

Signals that stop the rot: Regulation of secondary metabolite defences in cereals

“…Ca 2+ is one of the critical messenger ions and activates transcription factors, which regulate downstream gene expression. In sorghum, transcriptome analysis revealed that calcium signalling genes were upregulated as well as key biosynthetic genes for flavonoid phytoalexins in response to Bipolaris sorghicola [65]. Yang et al (2004) demonstrated that application of calcium channel blockers diminished the accumulation of avenanthramides, which led them to believe that calcium ions were critical to the activation of oat phytoalexin production [66].…”

“…However, this is not a definitive classification [97]. Global microarray analysis revealed that sorghum responds to exogenous SA and JA by up-regulating genes that form part of the phenylpropanoid pathway and generating 3-deoxyanthocyanidins, flavonoid phytoalexins [65]. Historically, based on studies in Arabidopsis, it has been thought that SA and JA act antagonistically; however, studies have both corroborated and contested this interaction in sorghum [24,35].…”

“…Interestingly, application of SA to sorghum and avirulent strains of F. graminearum to wheat heads resulted in the up-regulation of JA biosynthetic genes [65,98], thereby arguing that upon elicitation, plant hormones are synthesised de novo.…”