Expression of the fluorescent proteins DsRed and EGFP to visualize early events of colonization of the chickpea blight fungus Ascochyta rabiei

Abstract

Ascochyta blight caused by the ascomycete fungus Ascochyta rabiei, is a major biotic constraint of chickpea (Cicer arietinum L.), resulting in disastrous crop losses worldwide. To study early stages of development and pathogenic mechanisms of the fungus, two binary vectors for the constitutive expression of the red fluorescent protein (DsRed-Express) and the green fluorescent protein (EGFP1) were constructed. Furthermore, we have developed an improved and highly reproducible Agrobacterium tumefaciens-mediated transformation protocol for A. rabiei. Transformation events were confirmed through Southern hybridizations that suggest single-copy integration of reporter genes in majority of the transformants. High level expression of both DsRed and EGFP proteins was obtained both in spores and in mycelia as detected by fluorescence microscopy. Intense fluorescence was used as a highly efficient vital marker to visualize early developmental changes of the fungus. The formation of infection structures like appressoria and germ tubes were observed both in vitro and in planta. This work will be useful to develop methodologies for understanding the mechanisms of Ascochyta-chickpea interaction and functional genomics of A. rabiei towards the isolation of virulence genes.

Transcription Factor Repertoire of Necrotrophic Fungal Phytopathogen Ascochyta rabiei: Predominance of MYB Transcription Factors As Potential Regulators of Secretome

“…Though, with the recent advances in genomics, transcriptomics, and molecular studies of A. rabiei , it is now possible to carry out in depth investigations of A. rabiei pathogenicity. The genetic manipulations/transformations of A. rabiei have made identification of gene functions possible (Nizam et al, 2010). Similarly, various comprehensive studies of A. rabiei gene families have been performed in order to gain the molecular and evolutionary insights (Nizam et al, 2014a,b; Kim et al, 2015).…”

Agrobacterium tumefaciens-mediated transformation of Lasiodiplodia theobromae, the causal agent of gummosis in cashew nut plants

“…A. tumefaciens-T-DNA transfer has been extensively proven to be an efficient transformation system for filamentous fungi (Nizam et al, 2010;Vieira and Camilo, 2011;Maruthachalam et al, 2012). The binary vector pPm43GW-GFP-HPH, containing cassette with E. coli hph-resistance gene under the regulation of the Aspergillus nidulans trpC promoter and the PtGFP cassette containing the promoter toxA-5'-UTR from Pyrenophora tritici-repentis, was successfully employed.…”

“…The gfp gene encoding the green fluorescent protein (GFP) is widely employed as a fluorescence marker in many different filamentous fungus transformation protocols. Such protocols aim to image live-cell components during colonization or infecting processes (Nizam et al, 2010), to observe the dynamics of protein secretion in vivo (Ward, 2012), to test the function of cloned genes, to tag and clone pathogenicity genes, and to disrupt undesirable genes through insertional mutagenesis (Casas-Flores et al, 2004).…”

Development of a GFP-expressing Ustilaginoidea virens strain to study fungal invasion and colonization in rice spikelets

“…Previously, a variety of transformation systems have been employed to transform different filamentous fungi; like restriction enzyme mediated integration (REMI) (Sanchez et al, 1998;Kaufman et al, 2004;Kim et al, 2004), polyethylene glycol/CaCl 2 (Fitzgerald et al, 2003;Liu and Friesen, 2012), electroporation (Kuo et al, 2004;Turgeon et al, 2010;Tanaka et al, 2011), particle bombardment (Davidson et al, 2000;Hazell et al, 2000) and Agrobacterium tumefaciens mediated transformation (ATMT) (Michielse et al, 2005;Nizam et al, 2010;Cheng et al, 2012;Park et al, 2013).…”

“…For a successful ATMT of a fungus similar virulence genes that are utilized for plant transformation are required (Bundock et al, 1995;Michielse et al, 2004). Agrobacterium tumefaciens has an advantage over other methods because it can easily transform fungal strains through non-homologous recombination and T-DNA insertion at random sites in a single copy (Chen et al, 2000;Grimaldi et al, 2005;Wang et al, 2008;Nizam et al, 2010;Ding et al, 2011). In addition, ATMT also increases frequency of homologous recombination, a feature which is favorable for efficient target gene knock-out Zwiers and De Waard, 2001;Dobinson et al, 2004;Kim et al, 2011) and it also works well with several intact fungal structures (e.g., spores, mycelia, gill structures from mushroom), offering an alternative means for transforming fungi that do not readily produce protoplasts.…”