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Discovering and characterising the genetic mechanism controlling the resistance response to Ascochyta blight (Ascochyta rabiei) in chickpea (Cicer arietinum)

Tristan Coram1 and Edwin Pang2

1 Department of Biotechnology and Environmental Biology, RMIT University, Vic, 3083, Australia. Email s9806468@student.rmit.edu.au
2
Department of Biotechnology and Environmental Biology, RMIT University, Vic, 3083, Australia. Email eddie.pang@rmit.edu.au

Abstract

A chickpea accession highly resistant to ascochyta blight (ICC3996) was utilised for the construction of an enriched cDNA library. Enrichment for defence-related transcripts was achieved by harvesting chickpea tissue that had been challenged with A. rabiei spores. Random clones from this library were sequenced, resulting in a library of 1012 ESTs that were characterised according to their putative functions. Twenty-six ESTs were found to be directly associated with plant defence, whilst 450 showed no homology to existing GenBank, SwissProt or SpTrEMBL entries. The EST library provided a preliminary indication of defence-related gene expression in chickpea, and was used in subsequent microarray studies to uncover the specific genes and pathway involved in ascochyta blight resistance.

Media Summary

Gene expression studies of a chickpea accession highly resistant to Ascochyta blight may reveal the genetic basis for resistance.

Key Words

Ascochyta rabiei, Cicer arietinum, ESTs, resistance, microarray

Introduction

Chickpea (Cicer arietinum L.) is the third most important pulse crop in the world, and Australia is currently the largest exporter and sixth largest producer (Food and Agriculture Organisation 2004). A major factor limiting world chickpea production is a severe and destructive fungal disease known as Ascochyta blight (Ascochyta rabiei (Pass.) L.). Cultivated chickpeas do not possess an effective level of A. rabiei resistance (Akem 1999), prompting a search for resistance in other sources, such as uncultivated chickpea accessions. Studies of one uncultivated accession, ICC3996, have revealed a strong capacity for A. rabiei resistance (C. Pittock pers. comm.; Collard et al. 2001) in comparison to susceptible varieties such as Lasseter. Consequently, ICC3996 may be a valuable source of resistance (R) and defence-related genes that could be employed in the development of highly resistant chickpea varieties.

Considering that no definitive information exists on the function and number of genes controlling the pathway of A. rabiei resistance in chickpea, a more powerful method of defence-associated gene analysis is needed. Expressed Sequence Tag (EST) and microarray gene expression analysis appear to possess the capabilities to identify all genes involved in A. rabiei resistance, but in order to perform such analyses an extensive library of chickpea gene sequences is required. This paper reports the preliminary analysis of an enriched cDNA library synthesised from the highly resistant ICC3996 accession after inoculation with A. rabiei spores, and subsequent microarray gene expression studies.

Methods

An enriched cDNA library of ICC3996 was constructed by pooling 24 h and 48 h post-inoculation stem and leaf samples. Total RNA was extracted from the pooled sample using the RNeasy Plant Mini Kit (QIAGEN™). The cDNA library was constructed using the SMART™ PCR cDNA Synthesis Kit (Clontech™), where the resulting cDNA was ligated into pGEM-T Easy Vector (Promega™) and transformed into E. coli JM109 cells (Promega™).

Plasmid DNA was isolated from single colonies using the QIAquick Spin Miniprep Kit (QIAGEN™). Over 1000 clones were randomly selected and the quality of each insert was assessed by specific PCR amplification with T7 and SP6 primers. All clones were subjected to single-pass sequencing from the 5’ end of the vector using BigDye™ Terminator chemistry (PE Biosystems). Resulting ESTs were identified and characterised using BlastN and BlastX to determine sequence homology with existing entries in GenBank, SwissProt and SpTrEMBL.

Printing of microarray slides and subsequent hybridisation experiments were commenced at the time of writing, performed at the Australian Genome Research Facility (AGRF).

Results

Based on the knowledge that the minimum size of a functional gene-encoding region (exon) is about 200 bp, only plasmid inserts >200 bp were sequenced (Glick and Pasternak 1998). 1198 clones were single-pass sequenced, but 176 (17%) did not provide reliable sequence reads, resulting in a cDNA library consisting of 1012 ESTs. Sequence analysis revealed that 562 (56%) showed substantial homology to existing entries from either GenBank, SwissProt or SpTrEMBL, whilst 450 (44%) exhibited no significant sequence or functional homology. Consequently, all ESTs were grouped into general metabolic and functional categories (Figure 1).

With respect to A. rabiei resistance, the most interesting ESTs were those of the ‘defence’ category. Twenty-six ESTs were functionally associated with plant defence, but due to some redundancy only 21 represented unique sequences (Table 1).

The enrichment for defence-related ESTs appeared successful, especially considering that only 0.06% of genes in the Arabidopsis thaliana genome are involved in defence compared to 3% in this case (Gene Ontology Consortium 2000). The very large ‘unknown’ category may also contain novel chickpea R genes, and the ‘signalling’ category may contain ESTs essential to the coordination of defence responses. Consequently, the printed microarray slides incorporated all of these ESTs in an attempt to uncover the chickpea resistance mechanism. These expression experiments, commenced at the time of writing, were aimed at studying EST up- or down-regulation in a range of resistant and susceptible chickpea accessions, so that it may be possible to identify the genes involved and the pathway of A. rabiei resistance.

Although this study focused on defence-related ESTs, the remaining sequences may prove useful in the breeding chickpeas for agronomic characteristics such as seed quality, or for stress tolerances including cold and drought. Additionally, defence-related sequences may be used in molecular mapping relative to QTLs for A. rabiei resistance.

Figure 1. Functional classification of the 1012 Cicer arietinum (ICC3996) ESTs.

Table 1. Cicer arietinum (ICC3996) defence-related ESTs with sequence homology or similarity to GenBank, SwissProt or SpTrEMBL entries.

Clone Number

Nucleotide/Function Match

GenBank, SwissProt or SpTrEMBL Accession

E Value

Copy Number

CA0070

Extensin-like disease resistance protein

O82202

1e-28

1

CA0082

Gamma-thionen defensin/protease inhibitor

THGF_HELAN

2e-09

1

CA0188

Pathogen-induced translation initiation factor nps45

SUI1_BRAOL

2e-34

1

CA0191

S1-3 pathogen-induced protein

Q9MB24

2e-20

1

CA0221

Caffeoyl-CoA-methyltransferase

Q40313

2e-98

2

CA0228

Putative disease resistance protein

Q9LZ25

2e-09

1

CA0257

Transcription factor EREBP-1

Q9SE28

5e-07

3

CA0277

Avr9/Cf-9 rapidly elicited protein 65

Q9FQZ0

0.02

2

CA0303

Pathogenesis-related protein 4A

Q9M7D9

2e-61

1

CA0329

Beta-1-3-glucanase

E13B_PEA

3e-18

1

CA0442

Protein with leucine zipper

Q40156

1e-30

1

CA0452

Pathogen-induced transcriptional activator

Q9LL86

7e-11

1

CA0557

Leucine-zipper containing protein

Q945B7

8e-14

1

CA0632

Cinnamyl alcohol dehydrogenase (CAD1)

O65152

8e-35

1

CA0641

Nematode resistance protein Hs1pro-1

O04203

5e-06

1

CA0758

Multi-resistance protein

Q943U4

3e-12

1

CA0870

SNAKIN2 antimicrobial peptide precursor

Q93X17

2e-23

2

CA0984

Putative flavonol glucosyl transferase

Q9M156

2e-25

1

CA1079

Elicitor-induced receptor protein

Q9FH56

7e-06

1

CA1159

Pathogenesis-related protein class 10

PR1_MEDSA

5e-31

2

References

Akem C (1999) Ascochyta blight of chickpea: present status and future priorities. International Journal of Pest Management 45, 131-137.

Collard B, Ades P, Pang E, Brouwer J, Taylor P (2001) Prospecting for sources of resistance to ascochyta blight in wild Cicer species. Australasian Plant Pathology 30, 271-276.

Food and Agriculture Organisation (Accessed: 21 February 2004) FAO Statistical Databases – Agricultural Production. Updated: 3 February 2004 http://apps.fao.org/page/collections

Gene Ontology Consortium (2000) Gene ontology: tool for the unification of biology. Nature Genetics 25, 25-29.

Glick B, Pasternak J (1998) ‘Molecular Biotechnology: Principles and Applications of Recombinant DNA.’ American Society for Microbiology: Washington, D.C.

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