Functional Analysis of Autophagy Genes via Agrobacterium-Mediated Transformation in the Vascular Wilt Fungus Verticillium dahliae

Lei Zhou; Jun Zhao; Wangzhen Guo; Tianzhen Zhang

doi:10.1016/j.jgg.2013.04.006

Volume 40 Issue 8

Aug. 2013

Turn off MathJax

Article Contents

Article Navigation > Journal of Genetics and Genomics > 2013 > 40(8): 421-431

PDF( 2228 KB)

Functional Analysis of Autophagy Genes via Agrobacterium-Mediated Transformation in the Vascular Wilt Fungus Verticillium dahliae

doi: 10.1016/j.jgg.2013.04.006

National Key Laboratory of Crop Genetics and Germplasm Enhancement, Cotton Research Institute, Nanjing Agricultural University, Nanjing 210095, China

More Information

Corresponding author: E-mail address: cotton@njau.edu.cn (Tianzhen Zhang)
Received Date: 2013-01-23
Accepted Date: 2013-04-24
Rev Recd Date: 2013-04-22

Available Online: 2013-05-09

Publish Date: 2013-08-20

Abstract

Abstract

Autophagy is a widely conserved intracellular process for degradation and recycling of proteins, organelles and cytoplasm in eukaryotic organisms and is now emerging as an important process in foliar infection by many plant pathogenic fungi. However, the role of autophagy in soil-borne fungal physiology and infection biology is poorly understood. Here, we report the establishment of an Agrobacterium tumefaciens-mediated transformation (ATMT) system and its application to investigate two autophagy genes, VdATG8 and VdATG12, by means of targeted gene replacement and complementation. Transformation of a cotton-infecting Verticillium dahliae strain Vd8 with a novel binary vector pCOM led to the production of 384 geneticin-resistant transformants per 1 × 10⁶ conidia. V. dahliae mutants lacking either VdATG8 or VdATG12 exhibited reduced conidiation and impaired aerial hyphae production. Disease development on Arabidopsis plants was slightly delayed when inoculated with VdATG8 or VdATG12 gene deletion mutants, compared with the wild-type and gene complemented strains. Surprisingly, in vitro inoculation with unimpaired roots revealed that the abilities of root invasion were not affected in gene deletion mutants. These results indicate that autophagy is necessary for aerial hyphae development and plant colonization but not for root infection inV. dahliae.
- Autophagy,
- ATMT,
- Targeted gene replacement,
- Pathogenicity

FullText(HTML)

References(41)

References

[1]	Asakura, M., Ninomiya, S., Sugimoto, M. et al. Plant Cell, 21 (2009),pp. 1291-1304
[2]	Bartoszewska, M., Kiel, J.A. The role of macroautophagy in development of filamentous fungi Antioxid. Redox Signal., 14 (2011),pp. 2271-2287
[3]	Bishop, C.D., Cooper, R.M. An ultrastructural study of root invasion in three vascular wilt diseases Physiol. Plant Pathol., 22 (1983),pp. 15-27
[4]	Chen, P.Y., Wang, C.K., Soong, S.C. et al. Complete sequence of the binary vector pBI121 and its application in cloning T-DNA insertion from transgenic plants Mol. Breed., 11 (2003),pp. 287-293
[5]	Dobinson, K.F., Lecomte, N., Lazarovits, G. Can. J. Microbiol., 43 (1997),pp. 227-233
[6]	Dong, B., Liu, X.H., Lu, J.P. et al. Autophagy, 5 (2009),pp. 946-953
[7]	Fradin, E.F., Thomma, B.P. Mol. Plant Pathol., 7 (2006),pp. 71-86
[8]	Garas, N.A., Wilhem, S., Sagan, J.E. Phytopathology, 76 (1986),pp. 1005-1010
[9]	Geng, J.F., Klionsky, D.J. The Atg8 and Atg12 uniquitin-like conjugation systems in macroautophagy EMBO Rep., 9 (2008),pp. 859-864
[10]	Jeon, J., Park, S.Y., Chi, M.H. et al. Genome-wide functional analysis of pathogenicity genes in the rice blast fungus Nat. Genet., 39 (2007),pp. 561-565
[11]	Josefsen, L., Droce, A., Sondergaard, T.E. et al. Autophagy, 8 (2012),pp. 326-337
[12]	Kershaw, M.J., Talbot, N.J. Genome-wide functional analysis reveals that infection-associated fungal autophagy is necessary for rice blast disease Proc. Natl. Acad. Sci. USA, 106 (2009),pp. 15967-15972
[13]	Klosterman, S.J., Atallah, Z.K., Vallad, G.E. et al. Annu. Rev. Phytopathol., 47 (2009),pp. 39-62
[14]	Klosterman, S.J., Subbarao, K.V., Kang, S. et al. Comparative genomics yields insights into niche adaptation of plant vascular wilt pathogens PLoS Pathog., 7 (2011),p. e1002137
[15]	Liu, T.B., Liu, X.H., Lu, J.P. et al. Autophagy, 6 (2010),pp. 74-85
[16]	Liu, X.H., Lu, J.P., Zhang, L. et al. Eukaryot. Cell, 6 (2007),pp. 997-1005
[17]	Lu, J.P., Liu, X.H., Feng, X.X. et al. Curr. Genet., 55 (2009),pp. 461-473
[18]	Maruthachalam, K., Klosterman, S.J., Kang, S. et al. Mol. Biotechnol., 49 (2011),pp. 209-221
[19]	Michielse, C.B., Hooykaas, P.J., van den Hondel, C.A. et al. Curr. Genet., 48 (2005),pp. 1-17
[20]	Mullins, E.D., Chen, X., Romaine, P. et al. Phytopathology, 91 (2001),pp. 173-180
[21]	Nadal, M., Gold, S.E. Mol. Plant Pathol., 11 (2010),pp. 463-478
[22]	Nakayashiki, H., Hanada, S., Nguyen, B.Q. et al. RNA silencing as a tool for exploring gene function in ascomycete fungi Fungal Genet. Biol., 42 (2005),pp. 275-283
[23]	Nesher, I., Barhoom, S., Sharon, A. BMC Biol., 6 (2008),p. 9
[24]	O'Donnell, K., Cigelnik, E., Weber, N.S. et al. Phylogenetic relationships among ascomycetous truffles and the true and false morels inferred from 18S and 28S ribosomal DNA sequence analysis Mycologia, 89 (1997),pp. 48-65
[25]	Pinan-Lucarre, B., Balguerie, A., Clave, C. Eukaryot. Cell, 4 (2005),pp. 1765-1774
[26]	Rauyaree, P., Ospina-Giraldo, M.D., Kang, S. et al. Curr. Genet., 48 (2005),pp. 109-116
[27]	Reggiori, F., Klionsky, D.J. Autophagy in the eukaryotic cell Eukaryot. Cell, 1 (2002),pp. 11-21
[28]	Sambrook, J., Fritsch, E.F., Maniatis, T.
[29]	Sesma, A., Osbourn, A. The rice leaf blast pathogen undergoes developmental processes typical of root-infecting fungi Nature, 431 (2004),pp. 582-586
[30]	Thompson, J.D., Higgins, D.G., Gibson, T.J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice Nucleic Acids Res., 22 (1994),pp. 4673-4680
[31]	Tzima, A.K., Paplomatas, E.J., Rauyaree, P. et al. Fungal Genet. Biol., 47 (2010),pp. 406-415
[32]	Tzima, A.K., Paplomatas, E.J., Rauyaree, P. et al. Mol. Plant Microbe. Interact., 24 (2011),pp. 129-142
[33]	Tzima, A.K., Paplomatas, E.J., Tsitsigiannis, D.I. et al. Fungal Genet. Biol., 49 (2012),pp. 271-283
[34]	Vallad, G.E., Subbarao, K.V. Phytopathology, 98 (2008),pp. 871-885
[35]	Veneault-Fourrey, C., Barooah, M., Egan, M. et al. Autophagic fungal cell death is necessary for infection by the rice blast fungus Science, 312 (2006),pp. 580-583
[36]	Veronese, P., Narasimhan, M.L., Stevenson, R.A. et al. Plant J., 35 (2003),pp. 574-587
[37]	Wang, J.Y., Cai, Y., Gou, J.Y. et al. Appl. Environ. Microbiol., 70 (2004),pp. 4989-4995
[38]	Wilhelm, S. Phytopathology, 45 (1955),pp. 180-181
[39]	Xiao, C.L., Subbarao, K.V. Phytopathology, 88 (1998),pp. 1108-1115
[40]	Xie, Z., Klionsky, D.J. Autophagosome formation: core machinery and adaptations Nat. Cell Biol., 9 (2007),pp. 1102-1109
[41]	Yoshimoto, K., Takano, Y., Sakai, Y. Autophagy in plants and phytopathogens FEBS Lett., 584 (2010),pp. 1350-1358