Methods paperReal-time PCR monitoring of fungal development in Arabidopsis thaliana infected by Alternaria brassicicola and Botrytis cinerea
Introduction
Reliable methods for disease severity assessment are of crucial importance for physiological studies, either to address plant resistance against a pathogen or to monitor the aggressiveness of a pathogen strain. Most currently used assays rely on visual scoring of the symptoms, which is the most straightforward method but is confronted to several limitations. First, tests based on the arbitrary determination of discrete disease categories do not allow to perform elaborate statistical analysis of results. A very widespread alternative is to score either lesion diameter or conidia formation, but such tests are cumbersome and not well-suited to address early phases of fungal development. Biochemical tests like ergosterol or chitin quantification have also been used extensively, but they are not specific for a given pathogen and very often are not sufficiently sensitive [2], [9], [10].
In the case of Arabidopsis thaliana interacting either with Alternaria brassicicola or Botrytis cinerea, most currently used disease assessment protocols rely on lesion diameter or surface measurements (see e.g. [15], [20]). For Alternaria, however, this is not a satisfying parameter since the fungus does not grow beyond the inoculation spot on wild-type Arabidopsis plants. Hence, this technique is only useful to score dramatic phenotypes like the one exhibited by the pad mutants [15]. An alternative is to score the phenotype conferred by crossing the plants of interest with a mutant plant displaying a medium sensitivity to A. brassicicola, like pad3 (J.-P. Métraux, personal communication). However, unexpected epistatic relationships between the two mutations cannot be excluded and might lead to misinterpretations of the results. Indirect assessment of fungal biomass have also been used, like monitoring the expression of the UidA gene in transgenic fungal strains carrying this gene under the control of a strong constitutive promoter. However, such a technique is labour-intensive and quantification accurateness might sometimes be questioned [16]. In Botrytis-inoculated plants, lesion development is highly variable. Plant resistance tests are often based on lesion diameter measurement or on the proportion of inoculated leaves exhibiting spreading necrosis [12]. Direct assessment of fungal development on inoculated leaves might be an alternative to those techniques and provide more robust results.
Since its first application on the detection of Phytophtora strains in their host plants [3], real-time quantitative PCR has proven to be a very simple and reliable way to quantify the progression of numerous pathogens [11], [13], [14]. Compared to other classical diagnosis methods, real-time PCR-based assays are rapid, sensitive, specific and best suited to discriminate between slightly different levels of infection [19]. Moreover, since their specificity only rely on primer sequence, such assays are easy to develop and they can be transposed to virtually every pathosystem.
In this study, we aimed at developing a very sensitive assay to score disease progression on A. thaliana infected with the fungal pathogens A. brassicicola and B. cinerea, which would be suitable to compare wild-type and mutant plant behaviour toward those pathogens or to assess the aggressiveness of different fungal strains. We were also interested in a method that would allow to score fungal growth and plant defence reactions from the very beginning of the infection till its very late stages. Finally, our goal was to develop a very simple protocol that would be suitable for high throughput analysis.
Section snippets
Assay specificity, sensitivity and robustness
Usual real-time quantitative PCR controls were performed to check for the linearity of amplification over the dynamic range. Fig. 1 shows the regression curve obtained after amplification of serial dilutions of 6.25 pg to 16.7 ng A. brassicicola and B. cinerea genomic DNA with the CG9/CG10 and CG11/CG12 primers, respectively. Similar results were obtained when Arabidopsis genomic DNA was amplified with primers iASK1/iASK2, which are specific of Arabidopsis Shaggy-kinase-like gene At5g26751
Discussion
In this study, we developed a real-time PCR-based assay to follow disease progression on Arabidopsis plants infected with the fungi A. brassicicola and B. cinerea. It is based on the relative quantification of plant and fungal DNA in infected tissues by performing two real-time PCR reactions targeted at fungal and plant sequences on inoculated samples. Controls showed that the primer pairs used allow reliable DNA quantification over a very wide dynamic range (6.25 pg–16.7 ng DNA), providing an
Plant material and inoculation
Six to seven week-old A. thaliana (Columbia 0) plants grown under an 8 h photoperiod at 20 °C and 75% RH were used for inoculation. The A. brassicicola MUCL20297 strain was obtained from W.F. Broekaert (Catholic University of Leuven, Belgium). The B. cinerea strain was a kind gift from T. Barchietto (Biotransfer, Paris, France). Three 3 μl droplets of spore suspension were deposited on 4–5 mature leaves of each plant. Unless stated otherwise, the usual inoculums used were 5.104 and 5.105 sp/ml
Acknowledgments
The authors are very grateful to P. Mergaert (ISV-CNRS, Gif-sur-Yvette, France) and C. Levis (INRA Versailles, France) for stimulating discussions, to F.C. Küpper (The Scottish Association for Marine Science, Oban, UK) and M. Langlois-Meurinne (IBP, Orsay, France) for critical reading of the manuscript. They would like to acknowledge two anonymous reviewers who helped to improve this manuscript.
References (20)
- et al.
A critical assessment of the validity of ergosterol as an indicator of fungal biomass
Mycol. Res.
(1995) - et al.
Quantification of disease progression of microbial pathogens on Arabidopsis thaliana using real-time fluorescence PCR
FEMS Microbiol. Lett.
(2003) - et al.
An improved ergosterol assay to estimate fungal biomass in ectomycorrhizas
Mycol. Res.
(1990) - et al.
Disturbed correlation between fungal biomass and beta-glucuronidase activity in infections of Arabidopsis thaliana with transgenic Alternaria brassicicola
Plant Sci.
(1999) - et al.
Detection and quantification of Plectosphaerella cucumerina, a potential biological control agent of potato cyst nematodes, by using conventional PCR, real-time PCR, selective media, and baiting
Appl. Environ. Microbiol.
(2003) - et al.
Real-time quantitative PCR: DNA determination in isolated spores of the mycorrhizal fungus Glomus mossae and monitoring of Phytophthora infestans and Phytophthora citricola in their respective host plants
J. Phytopathol.
(1999) - et al.
Expression profiling of the whole Arabidopsis Shaggy-like kinase multigene family by real-time reverse transcriptase-polymerase chain reaction
Plant Physiol.
(2002) - et al.
Conventional PCR and real-time quantitative PCR detection of Helminthosporium solani in soil and on potato tubers
Eur. J. Plant Pathol.
(2001) - et al.
Abrogation of disease development in plants expressing animal antiapoptotic genes
Proc. Nat. Acad. Sci. USA
(2001) - et al.
A simple and rapid method for the preparation of plant genomic DNA for PCR analysis
Nucleic Acids Res.
(1991)
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