Fluorescent tagging of Verticillium species for assays of fungal adhesion to plant roots

disruption. The newly developed tools are not only used for Verticillium, but also for other filamentous fungi. These tools will allow characterizing quickly potential roles of putative adhesion genes from yeast complementation assays as well as putative Verticillium adhesin genes from the genome database analyses.

3.2.1. Fluorescent tagging of Verticillium species for assays of fungal adhesion to plant roots and for a new silencing system

3.2.1.1. Agrobacterium-mediated fungal transformation is simple and effective for gene transfer into genomes of Verticillium species

To facilitate genetic analysis of pathogenicity of Verticillium plant pathogens, two transformation methods including protoplast-mediated transformation and Agrobacterium tumefaciens-mediated transformation (ATMT) have been developed. Dobinson (1994) reported a DNA-mediated transformation system using protoplasts generated from V.

dahliae. Transformation efficiency of this method was quite low, only between 3 and 5 hygromycin resistant transformants/µg vector DNA. The DNA was integrated into the V.

dahliae genome with a single copy or multiple copies or even in tandem array (Dobinson, 1994). Therefore, this method is not used any longer for Verticillium transformation.

Currently, the most preferred method for Verticillium species is ATMT. The ATMT method has been successfully used for gene disruption, RNA-mediated gene silencing and fluorescence tagging in Verticillium species (Dobinson et al., 2004; Rauyaree et al., 2005;

Klimes et al., 2006; Eynck et al., 2007; Vallad and Subbarao, 2008; Singh et al., 2010;

Tzima et al., 2010; Gao et al., 2010; Knight et al., 2010; Paz et al., 2011).

In this work, we have improved the procedure for Verticillium transformation using A. tumefaciens as a DNA carrier. We used A. tumefaciens to transfer two fluorescent genes, GFP and DsRed, into two Verticillium pathogenic species (V. dahliae and V.

longisporum). A. tumefaciens bacterial cells were treated with MgCl2 and CaCl2 to become competent (see details in Materials and Methods). The bacterial competent cells can be preserved in glycerol at -80^oC for 1 year without any problem for transformation efficency.

The fluorescent gene-containing binary vectors were transformed separately into this bacterium by freezing the mixture (vector + bacterial cells) in liquid nitrogen, thawing it at 37^oC and recovering bacterial cells with antibiotic-free SOC medium. As a result, we could get about 100 positive colonies for 1 µg of each transforming vector. This method is

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effective and much easier to perform than electroporation method that requires special equipments (Singh et al., 2010).

To transfer the fluorescent gene (GFP or DsRed) from the binary vector in Agrobacterium to Verticillium, we mixed the fungal frozen spores with bacterial cells carrying the vector and spread the mixture on a filter paper of an induction medium plate containing acetosyringone (AS). During 72 hours of co-incubation at 25^oC, AS induces bacterial cells to deliver T-DNA (transfer DNA) fragment carrying the fluorescent gene from the binary vector to the fungal genome. Afterwards, the membrane was shifted to a selection plate for 10 days of additional incubation to favor the development of resistant transformants. On average, from about 1.5 million of the fungal frozen spores (0.15x10⁷) as material for transformation we obtained 40-50 hygromycin resistant transformants per plate. This method is much more efficient than the protoplast-mediated transformation that was reported by Dobinson (1994). The quality of Verticillium frozen spores is still good enough for ATMT method after 12 months of preservation at -80^oC, therefore ATMT requires less time than the protoplast-mediated method for material preparation of transformation.

3.2.1.2. Expression of the fluorescent genes DsRed and GFP in Verticillium plant pathogens

More than a decade, green fluorescent protein has been used popularly to light up fungal biology for exploring mechanisms of infection and interaction between host plants and fungal pathogens (Lorang et al. 2001; Sesma and Osbourn, 2004; Andrie et al., 2005;

Eynck et al., 2007; Vallad and Subbarao, 2008). In addition, the DsRed fluorescent protein is also a good reporter for ascomycetes fungi (Mikkelsen at al., 2003; Janus et al., 2007).

However, it has not been yet used for investigations of plant-Verticillium interaction. Here, we report that DsRed protein can be used fruitfully for this aspect. It can be also used to replace GFP protein if the green fluorescent signal is interfered by backgrounds from plant hosts.

We found that expression of red fluorescent gene DsRed under the control of gpdA promoter from Aspergillus nidulans promotes the red pigment formation in Verticillium species on solid media after 2-3 weeks at 25^oC. However, the red pigment is only visualized clearly on agar plates (PDA medium) containing hygromycin. At concentration of 50-100 µg/ml, hygromycin suppresses the formation of melanized survival structures,

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microsclerotia. This results in a reduction in black color of fungal mycelium and favours the clear appearance of the red color on agar plates (Figures 17A-C).

Figure 17. Expression of the DsRed fluorescent gene in Verticillium. (A) V. dahliae transformed with the empty vector pPK2-hph (left) and with the DsRed carrying vector pHQ1 (right). The transformants were grown on hygromycin-containing PDA plates for 2 weeks. (B) V. longisporum with empty vector pPK2-hph (left, white) and with DsRed carrying vector pHQ1 (right, red) after 4 weeks of growth on hygromycin-containing PDA medium. (C) Microsclerotia formation of V.

longisporum transformed with empty vector pPK2-hph (left) and with DsRed carrying vector pHQ1 (right) after 4 weeks of growth on PDA medium lacking hygromycin.

Similarly, GFP fluorescent gene under the control of ToxA promoter from fungal plant pathogen Pyrenophora tritici-repentis (Sesma and Osbourn, 2004) was also expressed strongly and effectively in both Verticillium species. However, expression of the fluorescent genes are very various among the tagged transformants. Therefore we recruited Southern hybridization to examine if the T-DNA copy number decided the signal strength.

The results showed that the fluorescent signal was not dependent of the DNA copy number integrated into the fungal genome (Figure 18).

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Figure 18. Correlation between the fluorescent signal and DNA copy number. Expression of DsRed fluorescent gene in V. dahliae strain Vd73 resulted in different accumulation of the red pigment in the fungal transformants. Southern hybridization with DsRed probe showed a single copy of T-DNA in most of transformants. The expression of the fluorescent genes seem to depend on position of T-DNA integration in the genome, but not on the DNA copy number.

3.2.1.3. Visualization of the early events of plant infection by Verticillium longisporum Germination, attachment and plant surface colonization of V. longisporum are the initial steps for a successful infection on oilseed rape (Eynck et al., 2007). We demonstrated that the early stages of plant infection by V. longisporum could be observed clearly using either the red fluorescent protein or green fluorescent protein (Figure 19).

Figure 19. The early infection stages of Arabidopsis through the roots by V. longisporum. The DsRed tagged V. longisporum (A) and GFP tagged V. longisporum (B) were used to infect A.

thaliana. From left to right: fungal germination of the roots, growth and penetration into the xylem, colonization of root surface and fungal growth inside the xylem vessle (photos: Michael Reusche).

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3.2.1.4. Dual-expression of GFP and DsRed in Verticillium species

Expression of two different fluorescent genes at the same time in Verticillum species using only one selection marker (hygromycin) would be helpful for testing new silencing (RNAi) systems. In these systems, one of the fluorescent genes can be co-silenced together with an endogenous gene, while the other is used as a marker to investigate the plant root infection ability of silenced mutants. We transformed the pHQ2 vector carrying GFP and DsRed genes into V. dahliae and V. longisporum. The resulting transformants resistant to hygromycin were selected for both the red and green fluorescent signals (Figure 20A). The stability of these fluorescent transformants were tested on the PDA medium lacking hygromycin for 5 successive generations using a single spore. Because silencing strategies are suitable for V. longisporum, a near diploid fungus, we performed some plant infection assays for a fluorescent version of this fungus (Vl43GR) using Arabidopsis thaliana and rapeseed Brassica napus (Figure 20B-C).

Figure 20. The dual-fluorescent tagging for V. longisporum and plant infection assays. (A) Co-expression of GFP and DsRed in V. longisporum Vl-43 resulting in the stable fluorescent version of this strain (Vl43GR). (B) The symptoms and the disease scores on Arabidopsis thaliana infected by V. longisporum. (C) The symptoms and the disease scores on rapeseed Brassica napus.

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The data indicated that the fluorescent version of V. longisporum (Vl43GR) could infect both plants with the same disease scores like the original strain Vl43. Therefore, this fluorescent strain could be used for further investigations instead of the original one. In addition, we also employed a stable dual fluorescent transformant of V. dahliae Vd73 (Vd73GR) to test efficiency of the new silencing system in parallel with the fluorescent V.

longisporum strain (Vl43GR).