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Identification of resistance-associated proteins in closely-related maize lines varying in aflatoxin accumulation

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Abstract

Aspergillus flavus infection of maize and subsequent contamination with carcinogenic aflatoxins poses serious health concerns, especially in developing countries. Maize lines resistant to A. flavus infection have been identified; however, the development of commercially-useful aflatoxin-resistant maize lines has been hindered due to a lack of breeding markers. To identify maize resistance-associated proteins (RAPs) as potential markers for breeding, 52 BC1S4 lines developed from crosses between five African maize inbreds and five temperate aflatoxin-resistant lines were screened using the kernel screening assay. Five pairs of closely-related lines that had 75–94% genetic similarity within each pair and which varied within each pair in aflatoxin accumulation were selected for proteomic investigation. Kernel embryo and endosperm protein profile differences within the pair and across pairs were compared using two-dimensional polyacrylamide gel electrophoresis. Differentially expressed (≥1.5-fold) RAPs were sequenced through tandem mass spectrometry and were identified as antifungal, stress-related, storage or regulatory proteins. Sequence homology analysis highlighted several proteins in maize that confer resistance to A. flavus infection and/or aflatoxin production.

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Abbreviations

2D:

Two-dimensional

COR:

Cold regulated

CV:

Coefficient of variation

ESI–MS/MS:

Electron spray ionization tandem mass spectrometry

EST:

Expressed sequence tag

GLX:

Glyoxalase

HSP:

Heat-shock protein

IITA:

International Institute of Tropical Agriculture

KSA:

Kernel screening assay

MALDI-TOF:

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

PAGE:

Polyacrylamide gel electrophoresis

RAPs:

Resistance associated proteins

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Acknowledgments

We thank Drs. Brian Scully and Marilyn Warburton for critical reviewing of the manuscript and Dr. Yurong Xie for technical assistance. Protein peptide sequencing using MALDI-TOF and ESI–MS/MS was performed at Protein Chemistry Core Facility, Baylor College of Medicine (Houston, TX, USA). This study was supported by USDA Cooperative Agreement 58-6435-6-055, the USDA-ARS Aflatoxin Elimination Workshop, and USDA National Research Initiative Competitive Grant 2002-35201-12541.

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Authors and Affiliations

  1. Department of Plant Pathology and Crop Physiology, Louisiana State University Agricultural Center, Baton Rouge, LA, 70803, USA

    Zhi-Yuan Chen

  2. Southern Regional Research Center, USDA-ARS, New Orleans, LA, 70179, USA

    Robert L. Brown & Thomas E. Cleveland

  3. International Institute of Tropical Agriculture, Ibadan, Nigeria

    Abebe Menkir

Authors
  1. Zhi-Yuan Chen
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  2. Robert L. Brown
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  3. Abebe Menkir
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  4. Thomas E. Cleveland
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Corresponding author

Correspondence to Robert L. Brown.

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11032_2011_9597_MOESM1_ESM.tif

Fig. S1 Protein spots differentially expressed in the endosperm tissue between resistant (on the left) and susceptible (on the right) closely-related maize lines. a spot 992, which was upregulated in the resistant line of all three pairs; b spot 1057, which was upregulated in the resistant line of two pairs; and c spot 1356, which was down-regulated in the resistant line of one pair, missing or did not change significantly in the other two pairs (TIFF 4979 kb)

11032_2011_9597_MOESM2_ESM.doc

Fig. S2 Sequence homology comparison (a) and a cladogram tree (b) showing relative similarities of peptide sequences from spots 436/473 and 1058 from embryo and spot 945 from endosperm to the partial amino acid sequences of globulin or cubin-domain containing proteins from different plant species. U47078, a globulin-like protein from Daucus carota (Dc); NM_128409 and ACG25229, a cupin family protein from Arabidopsis thaliana (At) and Zea mays (Zm), respectively; EF091701, a 7S globulin precursor from Ficus pumila (Fp); CD443502 and AW065979, two maize endosperm cDNA sequences. The mismatched amino acid residue proline (P) from spot 436/473 is underlined, and the amino acid residues that are identical or highly conserved among the species are indicated with “*” and “.”, respectively (DOC 64 kb)

11032_2011_9597_MOESM3_ESM.doc

Fig. S3 Peptide sequence alignment of spots 384 and 546 from embryo with glyoxalase family proteins from different plant species. ACG23936, glyoxalase family protein from Zea mays (Zm); ABF95269 and AB017042 are glyoxalase family proteins from Oryza sativa (Os) (Japonica Group); AAG48841, putative receptor serine/threonine kinase from Arabidopsis thaliana (At); AY241545, a glyoxalase I (GLXI) from Zea mays reported in our earlier study (Chen et al. 2004) (DOC 29 kb)

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Chen, ZY., Brown, R.L., Menkir, A. et al. Identification of resistance-associated proteins in closely-related maize lines varying in aflatoxin accumulation. Mol Breeding 30, 53–68 (2012). https://doi.org/10.1007/s11032-011-9597-3

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