Abstract
Red rot of sugarcane caused by the hemi-biotrophic fungal pathogen, Colletotrichum falcatum, is a major threat to sugarcane cultivation in many tropical countries such as India, Bangladesh, and Pakistan. With the accumulating information on pathogenicity determinants, namely, effectors and pathogen-associated molecular patterns (PAMPs) of C. falcatum, it is of paramount importance to decipher the functional role of these molecular players that may ultimately decide upon the outcome of sugarcane-C. falcatum interaction. Since C. falcatum is a multinucleated filamentous fungus, the conventional Agrobacterium-mediated transformation method could not be effectively utilized for targeted manipulation of genomic DNA. Hence, we developed a highly efficient protoplast-based transformation method for the virulent pathotype of C. falcatum — Cf671, which involves isolation of protoplast, polyethylene glycol (PEG)-mediated transformation, and regeneration of transformed protoplasts into hyphal colonies. In this study, germinating conidiospores of Cf671 were treated with different enzyme-osmoticum combinations, out of which 20 mg/mL lysing enzyme with 5 mg/mL β-glucanase in an osmoticum of 1.2 mol/L MgSO4 yielded maximum number of viable protoplasts. The resultant protoplasts were transformed with pAsp shuttle vector. Transformed protoplasts were regenerated into hyphal colonies under hygromycin selection and observed for GFP fluorescence. This protocol resulted in a transformation efficiency of > 130 transformants per μg of plasmid DNA. This method of transformation is rapid, simple, and more efficient for gene knockout, site-directed mutagenesis, ectopic expression, and other genetic functional characterization experiments in C. falcatum, even with large vectors (> 10 kb) and can also be applied for other filamentous fungi.
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The data that supports the findings of this study are available in the supplementary material of this article.
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Abbreviations
- PEG:
-
Polyethylene glycol
- ATMT:
-
Agrobacterium tumefaciens-Mediated transformation
- PMT:
-
Protoplast-mediated transformation
- TrpC:
-
Tryptophan synthase
- STC:
-
Sorbitol (glucitol)-Tris HCl-CaCl2
- gpdA:
-
Glyceraldehyde-3-phosphate dehydrogenase
References
Armesto C, Maia FGM, de Abreu MS et al (2012) Genetic transformation with the gfp gene of Colletotrichum gloeosporioides isolates from coffee with blister spot. Brazilian J Microbiol 43:1222–1229. https://doi.org/10.1590/S1517-83822012000300050
Ashwin NMR, Barnabas EL, Ramesh Sundar A et al (2017a) Disease suppressive effects of resistance-inducing agents against red rot of sugarcane. Eur J Plant Pathol 149:285–297. https://doi.org/10.1007/s10658-017-1181-1
Ashwin NMR, Barnabas L, Ramesh Sundar A et al (2017b) Comparative secretome analysis of Colletotrichum falcatum identifies a cerato-platanin protein (EPL1) as a potential pathogen-associated molecular pattern (PAMP) inducing systemic resistance in sugarcane. J Proteomics 169:2–20. https://doi.org/10.1016/j.jprot.2017.05.020
Ashwin NMR, Barnabas L, Ramesh Sundar A et al (2018) CfPDIP1, a novel secreted protein of Colletotrichum falcatum, elicits defense responses in sugarcane and triggers hypersensitive response in tobacco. Appl Microbiol Biotechnol 102:6001–6021. https://doi.org/10.1007/s00253-018-9009-2
Azizi M, Yakhchali B, Ghamarian A et al (2013) Cloning and expression of gumboro VP2 antigen in Aspergillus niger. Avicenna J Med Biotechnol 5:35–41
Casas-Flores S, Rosales-Saavedra Teresa, Herrera-Estrella A (2004) Three decades of fungal transformation: novel technologies. In: Balbás P, Lorence A (eds) Methods in molecular biology (Clifton, N.J.), 2nd edn. Humana Press Inc., Totowa, NJ, pp 315–325
Chakraborty BN, Patterson NA, Kapoor M (1991) An electroporation-based system for high-efficiency transformation of germinated conidia of filamentous fungi. Can J Microbiol 37:858–863. https://doi.org/10.1139/m91-147
Chung KR, Shilts T, Li W, Timmer LW (2002) Engineering a genetic transformation system for Colletotrichum acutatum, the causal fungus of lime anthracnose and postbloom fruit drop of citrus. FEMS Microbiol Lett 213:33–39. https://doi.org/10.1016/S0378-1097(02)00757-7
Díaz A, Villanueva P, Oliva V et al (2019) Genetic transformation of the filamentous fungus Pseudogymnoascus verrucosus of Antarctic origin. Front Microbiol 10:1–10. https://doi.org/10.3389/fmicb.2019.02675
Dufresne M, Bailey JA, Dron M, Langin T (1998) clk1, a serine/threonine protein kinase-encoding gene, is involved in pathogenicity of Colletotrichum lindemuthianum on common bean. Mol Plant-Microbe Interact 11:99–108. https://doi.org/10.1094/MPMI.1998.11.2.99
Epstein L, Lusnak K, Kaur S (1998) Transformation-mediated developmental mutants of Glomerella graminicola (Colletotrichum graminicola). Fungal Genet Biol 23:189–203. https://doi.org/10.1006/fgbi.1997.1029
Farina JI, Molina OE, Figueroa LIC (2004) Formation and regeneration of protoplasts in Sclerotium rolfsii ATCC 201126. J Appl Microbiol 96:254–262. https://doi.org/10.1046/j.1365-2672.2003.02145.x
Gao S, Yang Y, Wang C et al (2016) Transgenic sugarcane with a cry1Ac gene exhibited better phenotypic traits and enhanced resistance against sugarcane borer. PLoS ONE 11:1–16. https://doi.org/10.1371/journal.pone.0153929
Honeycutt RJ, Sobral BWS, Keim P, Irvine JE (1992) A rapid DNA extraction method for sugarcane and its relatives. Plant Mol Biol Report 10:66–72. https://doi.org/10.1007/BF02669266
Huang CN, Cornejo MJ, Bush DS, Jones RL (1986) Estimating viability of plant protoplasts using double and single staining. Protoplasma 135:80–87. https://doi.org/10.1007/BF01277001
Kachroo P, Potnis A, Chattoo BB (1997) Transformation of the rice blast fungusM agnaporthe grisea to benomyl resistance. World J Microbiol Biotechnol 13:185-187. https://doi.org/10.1023/A:1018589714357
Li D, Tang Y, Lin J, Cai W (2017) Methods for genetic transformation of filamentous fungi. Microb Cell Fact 16:1–13. https://doi.org/10.1186/s12934-017-0785-7
Madhavan A, Pandey A, Sukumaran RK (2017) Expression system for heterologous protein expression in the filamentous fungus Aspergillus unguis. Bioresour Technol 245:1334–1342. https://doi.org/10.1016/j.biortech.2017.05.140
Martin J.F. (2015) Fungal transformation: from protoplasts to targeted recombination systems. In: van den Berg M., Maruthachalam K. (eds) Genetic Transformation Systems in Fungi, 1st edn, Fungal Biology. Springer, Cham. pp https://doi.org/10.1007/978-3-319-10142-2_1
Meyer V, Mueller D, Strowing T, Stahl U (2003) Comparison of different transformation methods for Aspergillus giganteus. Curr Genet 43:371–377. https://doi.org/10.1007/s00294-003-0406-3
Michielse CB, Salim K, Ragas P et al (2004) Development of a system for integrative and stable transformation of the zygomycete Rhizopus oryzae by Agrobacterium-mediated DNA transfer. Mol Genet Genomics 271:499–510. https://doi.org/10.1007/s00438-004-1003-y
Nandakumar M, Malathi P, Sundar AR, Viswanathan R (2020) Use of green fluorescent protein expressing Colletotrichum falcatum, the red rot pathogen for precise host–pathogen interaction studies in sugarcane. Sugar Tech 22:112–121. https://doi.org/10.1007/s12355-019-00751-8
O’Connell R, Herbert C, Sreenivasaprasad S et al (2004) A novel Arabidopsis-Colletotrichum pathosystem for the molecular dissection of plant-fungal interactions. Mol Plant-Microbe Interact 17:272–282. https://doi.org/10.1094/MPMI.2004.17.3.272
Olmedo-Monfil V, Cortés-Penagos C, Herrera-Estrella A (2004) Three decades of fungal transformation: novel technologies. In: Balbás P, Lorence A (eds) Methods in molecular biology, 2nd edn. Humana Press Inc, Totowa, NJ, pp 315–325
Paietta JV (2015) Transformation of lithium acetate-treated Neurospora crassa. In: Berg MA van den, Maruthachalam K (eds) Genetic transformation systems in fungi, 1st edn, Fungal Biology. Springer, Cham. pp 193–197
Parisot D, Dufresne M, Veneault C et al (2002) clap1, a gene encoding a copper-transporting ATPase involved in the process of infection by the phytopathogenic fungus Colletotrichum lindemuthianum. Mol Genet Genomics 268:139–151. https://doi.org/10.1007/s00438-002-0744-8
Perfect SE, Hughes HB, O’Connell RJ, Green JR (1999) Colletotrichum: a model genus for studies on pathology and fungal-plant interactions. Fungal Genet Biol 27:186–198. https://doi.org/10.1006/fgbi.1999.1143
Poyedinok NL, Blume YB (2018) Advances, problems, and prospects of genetic transformation of fungi. Cytol Genet 52:139–154. https://doi.org/10.3103/S009545271802007X
Redman RS, Rodriguez RJ (1994) Factors affecting the efficient transformation of Colletotrichum species. Exp Mycol 18:230–246. https://doi.org/10.1006/emyc.1994.1023
Shin J-H, Han J-H, Park HH et al (2019) Optimization of polyethylene glycol-mediated transformation of the pepper anthracnose pathogen Colletotrichum scovillei to develop an applied genomics approach. Plant Pathol J 35:575–584. https://doi.org/10.5423/PPJ.OA.06.2019.0171
Stephenson SA, Hatfield J, Rusu AG et al (2000) CgDN3: an essential pathogenicity gene of Colletotrichum gloeosporioides necessary to avert a hypersensitive-like response in the host Stylosanthes guianensis. Mol Plant-Microbe Interact 13:929–941. https://doi.org/10.1094/MPMI.2000.13.9.929
Sundar RA, Ashwin N, Barnabas EL, et al (2015) Disease resistance in sugarcane — an overview. Sci Agrar Parana 14:200–212 https://doi.org/10.18188/1983-1471/sap.v14n4p200-212
van Leeuwen MR, Krijgsheld P, Bleichrodt R et al (2013) Germination of conidia of Aspergillus niger is accompanied by major changes in RNA profiles. Stud Mycol 74:59–70. https://doi.org/10.3114/sim0009
Viswanathan R (2018) Changing scenario of sugarcane diseases in India since introduction of hybrid cane varieties: path travelled for a century. J Sugarcane Res 8:1–35
Viswanathan R, Padmanaban P, Selvakumar R (2019) Emergence of new pathogenic variants in Colletotrichum falcatum, stalk infecting ascomycete in sugarcane: role of host varieties. Sugar Tech 22:473–484. https://doi.org/10.1007/s12355-019-00780-3
Zhou X, Li G, Xu JR (2011) Efficient approaches for generating GFP fusion and epitope-tagging constructs in filamentous fungi. In: Xu Jin-Rong and Bluhm Burton H. (eds) Methods in molecular biology (Methods and Protocols) Humana Press Inc., Totowa, NJ, pp 199–212. https://doi.org/10.1007/978-1-61779-040-9_15
Acknowledgements
The authors thank The Director, Indian Council of Agricultural Research—Sugarcane Breeding Institute, India for providing facilities and continuous encouragement.
Funding
The work was supported by the grant from Department of Biotechnology (DBT)—Government of India (Sanction no. BT/PR23621/BPA/118/297/2017). The first author received financial assistance from Council of Scientific and Industrial Research (CSIR)-New Delhi—Government of India (CSIR-UGC NET SRF, File no: 09/706(0002)/2017-EMR-I).
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Amalamol, D., Ashwin, N.M.R., Lakshana, K.V. et al. A highly efficient stratagem for protoplast isolation and genetic transformation in filamentous fungus Colletotrichum falcatum. Folia Microbiol 67, 479–490 (2022). https://doi.org/10.1007/s12223-022-00950-z
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DOI: https://doi.org/10.1007/s12223-022-00950-z