Bananas and plantains (Musa spp.), which are staple foods due to their high protein content and nutrition value as well as the main income source in many developing countries, are among the most important crops in the world. In fact, banana ranks as the fifth most important agricultural crop in world trade, making it the world’s leading fruit crop and a significant economic backbone to the export industry of many agriculture-based countries in Asia, Africa, and Latin America . Therefore, the global and local health of banana crops is of utmost importance to the world economy.
There are several devastating diseases that target the Musa crop . One such disease, Panama disease or Fusarium wilt, is caused by the fungus Fusarium oxysporum f. sp. cubense (Foc)  and is widely regarded as one of the most destructive plant diseases in the world. To date, Foc has devastated banana production and continues to threaten crops . The disease was first reported in 1874 in Australia and later destroyed the export trade based on the variety ‘Gros Michel’ by the 1950s. Since the 1960s, the resistant ‘Cavendish’ (AAA) subgroup of cultivars has dominated banana exports, becoming the major commercial variety in the world. However, an extremely virulent form of Foc, called ‘Tropical Race 4’ (Foc TR4), is capable of attacking the susceptible Cavendish variety, causing large losses in banana production in recent years .
Foc infects the lateral or feeder roots of banana plants upon contact . Foc infection causes wilt syndrome with the typical symptoms of necrosis and rotting of roots, rhizomes, and pseudostem vessels, which turn a reddish-brown/maroon color as the fungus grows through the tissues. After the decay of infected plants, the pathogen can survive in soil in chlamydospore form over a long period of time to infect other plants. Foc spores can spread through water or soil, and by adhering to vehicles and footwear. In the soil, Fusarium is difficult to control by general chemical measures, such as fungicides or soil fumigants . Therefore, resistance breeding is the preferred method of overcoming the Fusarium wilt of banana plants. However, because Cavendish bananas have a triploid (AAA) genome, they do not produce seeds, which hinders conventional breeding strategies .
Genetic engineering methods can improve the disease resistance of banana plants to Fusarium wilt ; however, little is known about the actual transcriptional changes and their regulation during the pathogen-plant interaction. Understanding the underlying changes during this interaction would allow for the identification of signal transduction pathways affected by infection and the interaction mechanisms during infection, which can lead to improvement of disease resistance of the banana plants. Traditional genome-wide analysis of gene expression of organisms under different conditions or, in the case of pathogens, at different life cycle stages, has mainly been carried out by microarrays, suppression subtractive hybridization (SSH), and cDNA-AFLP methods [9–12]. Van den Berg et al. (2007) used SSH and microarrays to show that cell wall-strengthening genes may be important for banana resistance to Fusarium wilt. However, the approach that was used suffers a number of drawbacks, including the fact that the genes are far from complete with only 79 clones . Recently, with the completion of banana genome sequence, a doubled haploid M. acuminata genotype (AA) has been shown to be highly resistant to Foc TR4 by phenotyping assays , but further research on its mechanism has not been performed, especially with relation to transcription. A resistant variety of the Cavendish banana (AAA) was acquired by somaclonal variation, using the RNA-seq and DGE methods, and it was discovered that recognition of PAMPs (pathogen-associated molecular pattern) and defense-related transcripts are involved in banana resistance to Foc TR4 infection . Therefore, elucidation of the mechanism by which Cavendish bananas respond to Foc TR4 infection is imperative.
One such promising method developed in recent years is next-generation sequencing, by which an enormous amount of sequence data can be rapidly obtained within a short period of time due to its high-throughput and high-coverage nature [15, 16]. RNA-Seq technology, which is based on deep-sequencing, enables more precise quantification of genome-wide transcript levels than previous, microarray-based methods . In this technology, whole mRNA or cDNA is mechanically fragmented for deep-sequencing, the results of which can be then mapped on a reference genome or used in de novo assembly to obtain a genome-wide transcriptome. Another method of great value for expression analysis is digital gene-expression (DGE) . DGE uses 17–21 base pair (bp) short fragments from the whole transcriptome as gene-specific tags and calculates the expression level of a gene from the frequency of its tag.
We previously used a green fluorescent protein (GFP)-tagged strain of Foc TR4 and characterized early events in infection and disease development of Cavendish plantlets . The combination of DGE and RNA-Seq allows us to easily perform transcriptome analysis without the need for an already-assembled reference genome. Despite the importance of the Foc pathogen for global banana production, RNA-Seq and DGE have not been used to investigate the main questions underlying the pathogen-banana interaction. Therefore, we aimed to investigate the changes in gene expression during Foc TR4 infection of banana roots using RNA-Seq and DGE analysis. For this purpose, we generated over 2.39 billion bases of high-quality DNA sequence and demonstrated the suitability of short-read sequencing for assembly and annotation of genes expressed in a triploid-genome plant without previous whole-genome information. We then identified 25,158 distinct sequences. Furthermore, we compared the gene expression profiles during an infection time course using DGE analysis. The assembled and annotated gene expression profiles provide an invaluable resource for the identification of differentially expressed genes during Foc TR4 infection of banana, which will enable us to screen for host susceptibility factors and to monitor shifts in Foc TR4 virulence.