Comparative analyses of genotype dependent expressed sequence tags and stress-responsive transcriptome of chickpea wilt illustrate predicted and unexpected genes and novel regulators of plant immunity

Abstract

Background: The ultimate phenome of any organism is modulated by regulated transcription of many genes. Characterization of genetic makeup is thus crucial for understanding the molecular basis of phenotypic diversity, evolution and response to intra- and extra-cellular stimuli. Chickpea is the world's third most important food legume grown in over 40 countries representing all the continents. Despite its importance in plant evolution, role in human nutrition and stress adaptation, very little ESTs and differential transcriptome data is available, let alone genotype-specific gene signatures. Present study focuses on Fusarium wilt responsive gene expression in chickpea.

Results: We report 6272 gene sequences of immune-response pathway that would provide genotype-dependent spatial information on the presence and relative abundance of each gene. The sequence assembly led to the identification of a CaUnigene set of 2013 transcripts comprising of 973 contigs and 1040 singletons, two-third of which represent new chickpea genes hitherto undiscovered. We identified 209 gene families and 262 genotype-specific SNPs. Further, several novel transcription regulators were identified indicating their possible role in immune response. The transcriptomic analysis revealed 649 non-cannonical genes besides many unexpected candidates with known biochemical functions, which have never been associated with pathostress-responsive transcriptome.

Conclusion: Our study establishes a comprehensive catalogue of the immune-responsive root transcriptome with insight into their identity and function. The development, detailed analysis of CaEST datasets and global gene expression by microarray provide new insight into the commonality and diversity of organ-specific immune-responsive transcript signatures and their regulated expression shaping the species specificity at genotype level. This is the first report on differential transcriptome of an unsequenced genome during vascular wilt.

Figure 1

Venn diagram depicting exclusive and overlapping CaESTs. The numbers signify ESTs specific to the susceptible and resistant genotypes and common between them.

Figure 2

Functional classification of CaUnigenes from root transcriptome. The genes identified were grouped into 23 functional classes as shown in the piechart based on metacyc, KOG and GO databases. The values represent the percentage of unigenes assigned to a particular functional class.

Figure 3

Comparative analysis of chickpea stress responsive genes with earlier known stress related genes. (A) Venn represents the overlap between ubiquitous, canonical and non canonical genes and the numbers signify unique and common stress responsive genes. (B) Expression pattern and prevalence of functional classes within the three groups. Each bar represents the number of genes in the respective functional class.

Figure 4

Tree view and functional categories of hierarchically clustered genes during Ca-Fusarium incompatible interaction. Tree view represents the expression patterns of genes displaying significant regulation in response to Fusarium infection at 24 hours post pathogen inoculation. Genes were organized using hierarchical clustering and displayed, including Cluster1 that represents the significantly repressed genes and Cluster2 that corresponds to significantly induced genes. Each row represents a single gene and each column corresponds to log2 (ratio) of an experimental sample. Black indicates the median level of expression, red indicates greater expression than the median and green less expression. Pie below each cluster shows the distribution of clustered genes into various functional categories. Detailed information on genes within each cluster can be found in additional file 6.

Figure 5

RNA blot analysis of few immunity induced genes of Ca-Fusarium incompatible interaction. 20 μg of total root RNA isolated from 25-day old chickpea seedlings were separated by 1.5% agarose gel. Lane U represents signal from the water treated control plants whereas lane 24 h represents RNA from 24 hours post pathogen challenge. The blots were hybridized with the respective gene specific probes as revealed by the microarray results.