Plants synthesize low-molecular-weight phytoalexins via secondary metabolic pathways to protect themselves from pathogens such as fungi [1
]. Phytoalexins of sorghum such as 3-deoxyanthocyanidins first appear in the cells under fungal attack, where they accumulate in cytoplasmic inclusion bodies. The inclusions migrate to the site of attempted penetration, become pigmented, and ultimately release phytoalexins to kill the fungus [2
]. Phytoalexins are produced mainly from aromatic amino acids (phenylalanine (Phe), tyrosine (Tyr)) by the action of many enzymes that sequentially catalyze biochemical reactions. Phenylalanine ammonia lyase (PAL) catalyzes the deamination of Phe to trans
-cinnamic acid; this is the first step in the biosynthesis of various phenylpropanoids, coumarins, flavonoids, and lignin [3
]. Aromatic-L-amino acid decarboxylase catalyzes decarboxylation of Tyr to tyramine; this is the first step in the production of isoquinoline alkaloids. Cytochrome P450s are diversified to make various phytoalexins and participants in metabolic networks, such as anthocyanins, tannins, flavones, and isoflavonoid [6
]. For phytoalexin synthesis, proper enzyme activity is required not only to synthesize the products needed, but also to avoid the accumulation of toxic metabolites.
Sorghum (Sorghum bicolor
L. Moench) is the fifth most commonly grown cereal in the world and is a rich source of sorghum-specific natural products. In response to pathogen infection, sorghum synthesizes a unique class of flavonoid phytoalexins, namely 3-deoxyanthocyanidins [2
]. These are structurally similar to anthocyanins except that they lack C-3 hydroxylation. As another example, sorghum seedlings accumulate high levels of dhurrin, a cyanogenic glycoside derived from tyrosine [9
]. Degradation of dhurrin releases hydrogen cyanide (HCN), which is very toxic to animals, plants, insects, and microorganisms [10
]. Dhurrin is also biologically important as a nitrogen storage compound. [12
]. Sorghum roots exude sorgoleone, a hydrophobic p
-benzoquinone compound that inhibits electron transfer in photosystem II to preclude competition for resources with neighboring plants [14
]. However, the enzymes required for phytoalexin synthesis have not been fully identified, and the nature of the coordinated gene expression for the production of these enzymes remains to be elucidated.
Target leaf spot is one of the major foliar diseases of sorghum under conditions of high humidity. This diseaseis caused by a necrotrophic fungus, Bipolaris sorghicola
]. Infected leaves of BTx623 have usually orange to red spots with straw-colored centers. Target leaf spot substantially reduces the production of plant biomass.
Studies of the functional genomics of sorghum began only after completion of the genomic sequence of sorghum BTx623 in 2009 [17
]. Sorghum has many proximally duplicated genes. For example, genes encoding cytochrome P450 enzymes are abundant in sorghum, with 326 copies, including the longest tandem gene array, of 15 genes [17
]. Each gene may be expressed under the conditions appropriate for catalyzing a particular biochemical reaction. However, the similarity of these genomic sequences makes it difficult to distinguish the expression of gene members of this family, even though various applications have been developed for detecting SNPs by using real-time quantitative reverse transcription – polymerase chain reaction (qRT-PCR) or microarray technology. Moreover, computational annotation has not yet fully covered whole genes. It is therefore important to identify whole transcripts (including unannotated transcripts) for complete gene expression profiling, and there is a need to develop technologies beyond arrays. Given the rapid progress of massive parallel sequencing technology, whole mRNA sequencing (mRNA-seq) has been used for gene expression profiling [18
]. A series of programs have been developed for building gene models directly based on the piling-up of short reads: the program Bowtie efficiently maps short reads on genomic sequences [23
], TopHat concatenates adjacent exons and identifies reads that bridge exon junctions [24
], and Cufflinks [25
] constructs gene models on the basis of the exons and bridging sequences predicted by Bowtie and TopHat. Thus, the use of sequencing-based expression profiling has the potential to overcome the limitations of PCR- or array-based profiling and can be used to identify key genes expressed among family members.
Here, from among duplicated genes we aimed to identify the key genes required for phytoalexin synthesis and to elucidate their coordinated expression in sorghum after infection with Bipolaris sorghicola. For this purpose, we performed whole mRNA sequencing by using massive parallel sequencing technology; differentially expressed genes, including unannotated genes, were identified on the basis of the piling-up of mapped reads. The differentially expressed genes were mapped on metabolic pathways; this analysis revealed their coordinated expression in primary metabolic networks to change the role of the TCA cycle and amino acids. We compared the expression of these genes with those of tandemly duplicated family genes in the sorghum genome and identified key enzymes in sorghum-specific phytoalexin synthesis. We also compared the genes with those located in the corresponding genomic regions in rice, and we discuss the evolutionary history of sorghum-specific phytoalexin synthesis. This work will help to elucidate the transcriptional regulation of primary and secondary metabolic pathways in response to pathogen infection in sorghum.