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  • Publication
    Accès libre
    Identification of genes expressed during the compatible interaction of grapevine with Plasmopara viticola through suppression subtractive hybridization (SSH)
    (2011)
    Legay, Guillaume
    ;
    Marouf, Elaheh
    ;
    Berger, Dave
    ;
    ; ;
    Slaughter, Ana R.
    Grapevine (Vitis vinifera) is the most widely cultivated and economically important fruit crop, but is susceptible to a large number of diseases. Downy mildew, caused by the obligate biotrophic oomycete pathogen Plasmopara viticola, is a common disease present in all regions where vines are cultivated. We used suppression subtractive hybridization (SSH) to generate two cDNA libraries enriched for transcripts induced and repressed, respectively, in the susceptible grapevine cultivar Chasselas 24 h after inoculation with P. viticola. Differential screening on glass slide microarrays yielded over 800 putative genes that were up-regulated in response to P. viticola infection and over 200 that were down-regulated. One hundred and ninety four of these, were sequenced, identified and functionally categorised. Transcript abundance of twelve genes over a 48 h time course was examined by reverse transcriptase quantitative real-time PCR (RT-qPCR). Ten of these genes were induced/enhanced by P. viticola challenge, confirming the results of the SSH. The vast majority of the genes identified are related to defence. Interestingly, many genes involved in photosynthesis were down-regulated.
  • Publication
    Accès libre
    Optimisation and comparison of transient expression methods to express the green fluorescent protein in the obligate biotrophic oomycete Plasmopara viticola
    (2008)
    Dubresson, Romain
    ;
    Kravchuk, Z.
    ;
    ;
    Grape downy mildew is caused by Plasmopara viti-cola, an obligate biotrophic oomycete and a major path-ogen of grapevine. Studying obligate biotrophic patho-gens is difficult as they cannot grow without their host. We therefore attempted to develop a method where the pathogen could be visualized and quantified in planta without killing the host plant. To this end P. viticola was transformed with the marker gene gfp coding for the green fluorescent protein. Various transformation methods, namely electroporation, particle bombard-ment and transformation with Agrobacterium tume-faciens were applied. Although some methods yielded positive transformation events, no stable strain of P. viticola expressing gfp could be generated. Using the electroporation method, we obtained transient P. viti-cola transformants expressing gfp over 4 generations. In contrast, particle bombardment failed in transform-ing P. viticola. Transformation with A. tumefaciens had a low efficiency, only some structures were fluorescent and fluorescence was never observed in the subsequent generations.
  • Publication
    Accès libre
    Beta-aminobutyric acid-induced resistance in grapevine against downy mildew: involvement of pterostilbene
    (Springer, 2008)
    Slaughter, Ana R.
    ;
    Hamiduzzaman, Mollah Md.
    ;
    Katia Gindro
    ;
    ;
    BABA, a non-protein amino acid, was used to induce resistance in grapevine against downy mildew. BABA-induced resistance was observed in the susceptible cv. Chasselas as well as in the resistant cv. Solaris. Following BABA treatment, sporulation of Plasmopara viticola was strongly reduced and the accumulation of stilbenes increased with time following infection. Induction of trans-piceide, trans-resveratrol and, more importantly, of trans-ɛ- and trans-δ-viniferin and trans-pterostilbene was observed in BABA-primed Chasselas. On the other hand, induction of trans-resveratrol, trans δ-viniferin and trans-pterostilbene was observed in BABA-primed Solaris. The accumulation of stilbenes in BABA-primed Solaris was much higher than that found in BABA-primed Chasselas. Furthermore, BABA-treatment of Solaris led to a rapid increase in transcript levels of three genes involved in the phenylpropanoid pathway: phenylalanine ammonia lyase, cinnamate-4-hydroxylase and stilbene synthase. BABA-primed Chasselas showed increased transcript levels for cinnamate-4-hydroxylase and stilbene synthase. Here we show that pre-treatment of a susceptible grapevine cultivar with BABA prior to infection with P. viticola primed the accumulation of specific phytoalexins that are undetectable in non-BABA-primed plants. As a result, the susceptible cultivar became more resistant to downy mildew.