A set of 20 genes which exhibit signaling potential were selected from a group of genes shown to be significantly up-regulated in B. cinerea infected A. thaliana leaf tissue 24 hours post infection (hpi). Two independent T-DNA lines were ordered from the European Arabidopsis Stock Centre (NASC) for each of these 20 genes. 29 homozygous T-DNA lines have been isolated with at least one available for each of the 20 chosen genes. A T-DNA insertion particularly within exons or 5’UTR typically results in gene disruption such that a homozygous T-DNA line is an effective knockout line for the gene in question (see Fig. 2).
Figure 2. A shows the two possible alleles present in T-DNA lines which need to be distinguished. Binding position of screening primers are also shown. B Shows expected PCR products on a gel for each potential genotype, homozygous wild type (HM WT), heterozygous (HT) and homozygous T-DNA knockout (HM T-DNA).
Screening T-DNA knockout lines for susceptibility to B. cinerea is done using a droplet inoculation method. Here detached leaves are inoculated with 10µl droplets of grape juice containing 1 x 105 spores/ml. Lesion perimeters where then measured 72hpi and compared to lesions on wild type leaves. A T-DNA knockout for each of the 20 genes has been screened in this manner but none seem to have given any noticeable phenotype comparable to the positive control (BOS1) being used (Fig 3).
Figure 3 Shows wild type (WT) and Botrytis susceptible (BOS1) mutant leaves inoculated with B. cinerea spores in grape juice compared and wild type mock inoculated with sterile grape juice (-ve). Photographs are taken at 24 hour intervals post infection.