Armillaria mellea, the causal agent of grapevine root rot, induces a set of defense genes in grapevine roots

Grapevine root rot, caused by the fungus Armillaria mellea, is a serious and increasing disease in some important grape growing areas such as California and North East of Italy (Baumgartner & Rizzo, 2002; De Luca et al., 2003). A. mellea infects grape roots and produces extensive fans of hyphae that spread under the bark decaying the underlying xylem. Infected plants show a decline in vigor, stunted shoots and leaves, dwarfed bunches and autumnal early change in leaf color (De Luca et al., 2003). Diseased plants normally die some years after pathogen infection. Since commercial pesticides are not effective to control Armillaria spp. infections (Aguin et al., 2006) and resistant Vitis rootstocks have not been identified (De Luca et al., 2003), the development of efficient control methods would be important to prevent the diffusion of the pathogen. In pot trials, young grapevines (2 years old) start to show symptoms of A. mellea infection and hyphal root penetration only from the second year after pathogen inoculation (Prodorutti et al 2009). These observations suggest the existence in young roots of a tolerance mechanism which delays root rot progress which might help in the development of new disease-control approaches. In the present study we dissected at molecular level the early defense response to A. mellea of the root of young plants from a widely used grapevine rootstock (Kober 5BB), by identification of the genes modulated 24 h after A. mellea inoculation and by assaying the antifungal activity of the proteins encoded by these genes. A subtracted cDNA library was constructed using a suppression subtractive hybridization (SSH) approach. The subtraction was done in order to enrich the library for cDNA sequences present in the A. mellea–inoculated Kober 5BB roots and absent from the untreated control roots. In total, 2,226 clones were collected and subjected to validation screening by hybridization. Following this check, 56 clones were considered truly up-regulated and were subjected to single pass sequencing, quality check, and clustering. This step yielded 24 unique sequences deposited in the public database. Functional annotation of the sequences according to the Gene Ontology (GO) categories revealed that they were involved in defense response (n = 4), response to stress (n = 1), hormone metabolism (n = 1), signal transduction (n = 3), regulation of gene expression (n = 4), protein modification (n = 1), and cell wall organization (n = 3). To validate the SSH results, we measured the relative expression of 11 genes (Table 1) by RT-qPCR in the RNA samples used to construct the SSH library and in roots treated with A. mellea under greenhouse or in vitro conditions. These genes were selected because they were putatively related to plant defense response (PIN1, PIN2, Tr, TL, GST, ACO, TF, and CaM) or because they had never before been associated with this process (GLB, Pro-R, and PCr)