The Plant Pathology Journal

The Korean Society of Plant Pathology (한국식물병리학회)
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Construction of a High-Quality Yeast Two-Hybrid Library and Its Application in Identification of Interacting Proteins with Brn1 in Curvularia lunata

  • Gao, Jin-Xin (School of Agriculture and Biology, Shanghai Jiaotong University) ;
  • Jing, Jing (School of Agriculture and Biology, Shanghai Jiaotong University) ;
  • Yu, Chuan-Jin (School of Agriculture and Biology, Shanghai Jiaotong University) ;
  • Chen, Jie (School of Agriculture and Biology, Shanghai Jiaotong University)
  • Received : 2015.01.01
  • Accepted : 2015.04.07
  • Published : 2015.06.01
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Abstract

Curvularia lunata is an important maize foliar fungal pathogen that distributes widely in maize growing area in China, and several key pathogenic factors have been isolated. An yeast two-hybrid (Y2H) library is a very useful platform to further unravel novel pathogenic factors in C. lunata. To construct a high-quality full length-expression cDNA library from the C. lunata for application to pathogenesis-related protein-protein interaction screening, total RNA was extracted. The SMART (Switching Mechanism At 5' end of the RNA Transcript) technique was used for cDNA synthesis. Double-stranded cDNA was ligated into the pGADT7-Rec vector with Herring Testes Carrier DNA using homologous recombination method. The ligation mixture was transformed into competent yeast AH109 cells to construct the primary cDNA library. Eventually, a high qualitative library was successfully established according to an evaluation on quality. The transformation efficiency was about $6.39{\times}10^5$ transformants/$3{\mu}g$ pGADT7-Rec. The titer of the primary cDNA library was $2.5{\times}10^8cfu/mL$. The numbers for the cDNA library was $2.46{\times}10^5$. Randomly picked clones show that the recombination rate was 88.24%. Gel electrophoresis results indicated that the fragments ranged from 0.4 kb to 3.0 kb. Melanin synthesis protein Brn1 (1,3,8-hydroxynaphthalene reductase) was used as a "bait" to test the sufficiency of the Y2H library. As a result, a cDNA clone encoding VelB protein that was known to be involved in the regulation of diverse cellular processes, including control of secondary metabolism containing melanin and toxin production in many filamentous fungi was identified. Further study on the exact role of the VelB gene is underway.

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References

  1. Bayram, O. and Braus, G. H. 2012. Coordination of secondary metabolism and development in fungi: the velvet family of regulatory proteins. FEMS Microbiol. Rev. 36:1-21. https://doi.org/10.1111/j.1574-6976.2011.00285.x
  2. Bayram, O., Krappmann, S., Ni M., Bok, J. W., Helmstaedt, K., Valerius O., Braus-Stromeyer, S., Kwon, N. J., Keller, N. P., Yu, J. H. and Braus, G. H. 2008. VelB/VeA/LaeA complex coordinates light signal with fungal development and secondary metabolism. Science 320:1504-1506. https://doi.org/10.1126/science.1155888
  3. Bell, A. A. and Wheeler, M. H. 1986. Biosynthesis and function of fungal melanins. Annu. Rev. Phytopathol. 24:411-451. https://doi.org/10.1146/annurev.py.24.090186.002211
  4. Brakhage, A. A., Langfelder, K., Wanner, G., Schmidt, A. and Jahn, B. 1999. Pigment biosynthesis and virulence. Contrib. Microbiol. 2:205-215. https://doi.org/10.1159/000060296
  5. Butler, M. J. and Day, A.W. 1998. Fungal melanins: a review. Can. J. Microbiol. 44:1115-1136. https://doi.org/10.1139/w98-119
  6. Conrad, D. H., Goyette, J. and Thomas, P. S. 2008. Proteomics as a method for early detection of cancer: a review of proteomics, exhaled breath condensate, and lung cancer screening. J. Gen. Intern. Med. 23:78-84. https://doi.org/10.1007/s11606-007-0411-1
  7. Dai, F. C., Gao, W. D., Wu, R. J. and Jin, X. H. 1995. A noticeable corn disease: Curvularia leaf spot. ACTA Phytopathol. Sin. 25:330.
  8. Dai, F. C., Wang, X. M., Zhu, Z. D., Gao, W. D. and Huo, N. X. 1998. Curvularia leaf spot of maize: pathogens and varietal resistance. ACTA Phytopathol. Sin. 2:123-129.
  9. Feng, J., Gao, Z., Xue, C., Zhuang, J., Chen, J. and Bai, S. 2002. The pathogenesis of the cell-degrading enzymes produced by Curvularia lunata. Rain Fed Crops 22:164-167.
  10. Gao, J. X., Jing, J., Liu, T. and Chen, J. 2015. Identification of Clt-1-regulated proteins associated with the production of non-host-specific toxin and pathogenicity in Cochliobolus lunatus. J. Phytopathol. 163:33-41. https://doi.org/10.1111/jph.12278
  11. Gao, J. X., Liu, T. and Chen, J. 2014a. Insertional mutagenesis and cloning of the gene required for the biosynthesis of the non-host specific toxin in Cochliobolus lunatus that causes maize leaf spot. Phytopathology 104:332-339. https://doi.org/10.1094/PHYTO-07-13-0190-R
  12. Gao, S. G., Li, Y. Q., Gao, J. X., Suo, Y. J., Fu, K. H., Li, Y. Y. and Chen, J. 2014b. Genome sequence and virulence variation-related transcriptome profiles of Curvularia lunata, an important maize pathogenic fungus. BMC Genomics 15:627. https://doi.org/10.1186/1471-2164-15-627
  13. Gao, S. G., Zhou, F. H., Liu, T., Li, Y. Y. and Chen, J. 2012. A MAP kinase gene, Clk1, is required for conidiation and pathogenicity in the phytopathogenic fungus Curvularia lunata. J. Basic. Microbiol. 53:214-223.
  14. Gubler, U. and Hoffman, B. J. 1983. A simple and very efficient method for generating cDNA libraries. Gene 25:263-269. https://doi.org/10.1016/0378-1119(83)90230-5
  15. Han, X. F., Luo, J., Wu, N., Matand, K., Yang, B. J., Wu, H. J., Zhang, L. J. and Wang, H. B. 2008. Construction and characterization of a goat mammary gland cDNA library. Mol. Biotechnol. 38:187-193. https://doi.org/10.1007/s12033-007-9020-9
  16. Jiang, J. H., Yun, Y. Z., Liu, Y. and Ma, Z. H. 2012. FgVELB is associated with vegetative differentiation, secondary metabolism and virulence in Fusarium graminearum. Fungal Genet. Biol. 49:653-662. https://doi.org/10.1016/j.fgb.2012.06.005
  17. Kawamura, C., Tsujimoto, T. and Tsuge, T. 1999. Targeted distribution of a melanin biosynthesis gene affects conidial development and UV Tolerance in the Japanese Pear pathotype of Aternaria alternata. Mol. Plant-Microbe Interact. 12:59-63. https://doi.org/10.1094/MPMI.1999.12.1.59
  18. Kulpa, D., Topping, R. and Telesnitsky, A. 1997. Determination of the site of first strand transfer during Moloney murine leukemia virus reverse transcription and identification of strand transfer-associated reverse transcriptase errors. EMBO J. 16:856-865. https://doi.org/10.1093/emboj/16.4.856
  19. Lee, L. Y., Wu, F. H., Hsu, C. T., Shen, S. C., Yeh, H. Y., Liao, D. C., Fang, M. J., Liu, N. T., Yen, Y. C. and Dokladal, L. 2012. Screening a cDNA library for protein-protein interactions directly in planta. Plant Cell 24:1746-1759. https://doi.org/10.1105/tpc.112.097998
  20. Liu, T., Liu, L., Jiang, X., Huang, X. and Chen, J. 2009. A new furanoid toxin produced by Curvularia lunata, the causal agent of maize Curvularia leaf spot. Can. J. Plant Pathol. 31:22-27. https://doi.org/10.1080/07060660909507568
  21. Liu, T., Xu, S., Liu, L., Zhou, F., Hou, J. and Chen, J. 2011. Cloning and characteristics of Brn1gene in Curvularia lunata causing leaf spot in maize. Eur. J. Plant Pathol. 131:211-219. https://doi.org/10.1007/s10658-011-9800-8
  22. Ma, J., Huang, X., Wang, X., Chen, X., Qu, Z., Huang, L. and Kang, Z. 2009. Identification of expressed genes during compatible interaction between stripe rust (Puccinia striiformis) and wheat using a cDNA library. BMC genomics 10:586. https://doi.org/10.1186/1471-2164-10-586
  23. Ma, Y., Ruan, Q., Ji, Y., Wang, N., Li, M., Qi, Y., He, R., Sun, Z. and Ren, G. 2011. Novel transcripts of human cytomegalovirus clinical strain found by cDNA library screening. Genet. Mol. Res. 10:566-575. https://doi.org/10.4238/vol10-2gmr1059
  24. Macri, F. and Lenna, P. 1974. Leaf corn blight incited by Curvularia lunata (Wakk.) Boed. J. Plant Pathol. 10:27-35.
  25. McClelland, M., Nelson, M. and Raschke, E. 1994. Effect of sitespecific modification on restriction endonucleases and DNA modification methyltransferases. Nucleic Acids Res. 22:3640-3659. https://doi.org/10.1093/nar/22.17.3640
  26. Miller, J. and Stagljar, I. 2004. Using the yeast two-hybrid system to identify interacting proteins. Methods Mol. Biol. 261:247-262.
  27. Stynen, B., Tournu, H., Tavernier, J. and Van Dijck, P. 2012. Diversity in genetic in vivo methods for protein-protein interaction studies: from the yeast two-hybrid system to the mammalian split-luciferase system. Microbiol Mol. Biol. Rev. 76:331-382. https://doi.org/10.1128/MMBR.05021-11
  28. Wang, J. Y. and Chen, J. 2011. Cloning and functional analysis of Clm1 in Curvularia lunata. ACTA Phytopathol Sin. 41:464-472.
  29. Wang, Y. Y., Zhang, T., Zhou, Q. M. and Wei, J. C. 2011. Construction and characterization of a full-length cDNA library from mycobiont of Endocarpon pusillum (lichen-forming Ascomycota). World J. Microb. Biot. 27:2873-2884. https://doi.org/10.1007/s11274-011-0768-5
  30. Xu, S. F., Chen, J., Liu, L. X., Wang, X. F., Huang, X. L. and Zhai, Y. H. 2007. Proteomics associated with virulence differentiation of Curvularia lunata in maize in China. J. Integr. Plant Biol. 49:487-496. https://doi.org/10.1111/j.1744-7909.2007.00469.x
  31. Zhao, W. J., Zhang, H., Bo, X., Li, Y. and Fu, X. 2009. Generation and analysis of expressed sequence tags from a cDNA library of moniezia expansa. Mol. Biochem. Parasitol. 164:80-85. https://doi.org/10.1016/j.molbiopara.2008.11.009
  32. Zhu, Y. Y., Machleder, E. M., Chenchik, A., Li, R. and Siebert, P. D. 2001. Reverse transcriptase template switching: a SMART approach for full-length cDNA library construction. Biotechniques 30:892-897.

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