Primary metabolism of chickpea is the initial target of wound inducing early sensed Fusarium oxysporum f. sp. ciceri race I

Abstract

Background: Biotrophic interaction between host and pathogen induces generation of reactive oxygen species that leads to programmed cell death of the host tissue specifically encompassing the site of infection conferring resistance to the host. However, in the present study, biotrophic relationship between Fusarium oxysporum and chickpea provided some novel insights into the classical concepts of defense signaling and disease perception where ROS (reactive oxygen species) generation followed by hypersensitive responses determined the magnitude of susceptibility or resistant potentiality of the host.

Methodology/principal findings: Microscopic observations detected wound mediated in planta pathogenic establishment and its gradual progression within the host vascular tissue. cDNA-AFLP showed differential expression of many defense responsive elements. Real time expression profiling also validated the early recognition of the wound inducing pathogen by the host. The interplay between fungus and host activated changes in primary metabolism, which generated defense signals in the form of sugar molecules for combating pathogenic encounter.

Conclusions/significance: The present study showed the limitations of hypersensitive response mediated resistance, especially when foreign encounters involved the food production as well as the translocation machinery of the host. It was also predicted from the obtained results that hypersensitivity and active species generation failed to impart host defense in compatible interaction between chickpea and Fusarium. On the contrary, the defense related gene(s) played a critical role in conferring natural resistance to the resistant host. Thus, this study suggests that natural selection is the decisive factor for selecting and segregating out the suitable type of defense mechanism to be undertaken by the host without disturbing its normal metabolism, which could deviate from the known classical defense mechanisms.

Figure 1. Phenotypical changes of chickpea plants upon Fusarium oxysporum f.sp. ciceri (Race 1) attack.

Infected JG62 plants at 4DPI (a), 8DPI (b) and 12DPI(c). Infected WR315 plants at 4DPI (d), 8DPI (e) and 12DPI (f).

Figure 2. Sectional views of infected roots of chickpea plants stained with Trypan blue and lactophenol.

Root section of infected JG62 plants at 4DPI (a and b), 8DPI (c and d) and 12DPI (e and f). Bars represent 10 µm.

Figure 3. Scanning electron micrographs of infected roots of chickpea plants.

Root section of infected JG62 plants at 4 DPI, 8 DPI, 12 DPI showing tissue disintegration (a), (c), (e) and conidia (b), (d), (f), respectively. Root section of infected WR315 plants at 15DPI showing xylem vessels (g) and tissue damage with conidia at 28DPI (h).

Figure 4. cDNA-AFLP gel profile of non-infected and infected JG62 and WR315 plant samples using different primers.

Lanes 1, 5, 9 non-infected JG62; lanes 3,7,11 non-infected WR315; lanes 2, 6 and 10 infected JG62 and lanes 4, 8, 12 infected WR315. Primer combinations used were lanes 1 to 4, E-AGC/M-CAC; lanes 5 to 8, E-AGC/M-CAG and lanes 9 to 12 E-AGC/M-CAT. Arrows indicate some of the bands selected for further analysis.

Figure 5. Relative expression of early defense response genes.

Expression of ATPase E and F subunit, rapid alkalinization factor, serine threonine kinase and phopholipase C at 48, 72 and 96 hours post fungal induction in JG62 and WR315 plants. Error bars represent standard error (n = 3).

Figure 6. Relative expression of wound responsive genes.

Expression of arginase, isoflavanoid biosynthetic gene, cytochrome P450 monoxygenase, drought stress ESTs and DNA methylation sensitive gene fragment at 48, 72 and 96 hours post fungal induction in JG62 and WR315 plants. Bars represent standard error (n = 3).

Figure 7. Relative expressions of genes related to primary metabolism.

Expression of beta amylase, sucrose synthase, invertase, hydrolase, nitrate transporter, acyl activating enzyme, 14-3-3 related protein, plastid division regulator and sugar transporter at 48, 72 and 96 hours post fungal induction in JG62 and WR315 plants. Bars represent standard error (n = 3).

Figure 8. Relative expression of transcription regulators, structural and antifungal genes.

Expression of Ribosomal protein RPS6 and RPL34, armadillo beta catenin repeat like protein, tubulin folding cofactor, cytochrome oxidase subunit 1 (COX1) and cystatin at 48, 72 and 96 hours post fungal induction in JG62 and WR315 plants. Bars represent standard error (n = 3).

Figure 9. Schematic pathway predicting the role of pathogen induced genes in defense.

Integrated pathway map shows the role of pathogen induced defensive genes involved in early defense, wound response, primary metabolism, transcriptional regulation and antifungal activity. The ESTs are indicated in stars.