Increased tolerance to wheat Powdery Mildew by heterologous constitutive expression of the Solanum chacoense snakin-1 gene.

FACIO, P; VAZQUEZ- ROVERE, C; HOPP, EH; GONZALEZ, G; DECIMA, C; FAVRET, E; DIAZ PALEO, A; FRANZONE, P

St. Petersburg

Congreso; 8th International Wheat Conference; 2010

8th International Wheat Conference

Plant diseases constitute an important limitation to the world food production. At present and due to production costs, efficiency and protection of the environment, a considerable effort is devoted to investigate the use of transgenes capable of conferring significant levels of resistance to phytopathogenic fungi. In this context, the effect of constitutive expression of antimicrobial peptide snakin 1, derived from Solanum chacoense, to increased wheat resistance against fungal diseases was assayed after challenging transgenic wheat plants expressing the SN1 gene with the fungus Blumeria graminis f. sp. tritici which cause ?powdery mildew? disease. Snakin 1 peptides are very similar in both S. chacoense and S. tuberosum (potato) and are the main antifungal proteins present in tubers. For proper expression in monocots, the original intron had to be removed from the coding region of SN1 under the transcriptional control of the constitutive promoter of the maize ubiquitin gene and introduced in wheat genotypes ProINTA Federal, SH9826 and SH9856 through a biolistic transformation procedure. The bar gene was used as selectable marker under the control of the rice actin gene promoter. SN1 and bar genes present individually in two different plasmids, were co bombarded on scutella of wheat immature embryos. The presence of bar and SN-1 genes was detected by PCR and the copy number of the transgenes estimated by Southern blot non radioactive digoxigenin hybridisation. Transcript analyses were performed by RT-PCR and quantitative Real Time RT-PCR. Transformation experiments provided 41 primary transgenic plants (T0) expressing the SN1 transcript. Transgene expression in eight of these T0 plants was silenced in the T1 or T2 generations. Transgene expression remained stable through T2 and T3 generations derived from the rest of the T0 plants obtained. Transgenic plants of T2 and T3 progenies expressing the SN1 gene were evaluated against the phytopathogenic fungus B. graminis f. sp. tritici. Inoculation assays were carried out on detached primary leaves. Leaf segments of approximately 3 cm were placed in Petri dishes on water-agar medium supplemented with benzylaminopurine to delay leaf tissue senescence. Each Petri dish contained leaf samples of plants expressing SN1, plants non expressing SN1 and non transgenic plants (wild-type) used as control. Inoculation was carried out by shaking infected plants at the top of a cardboard pipe of 1 meter height. Four to five open Petri dishes were placed at the bottom of the pipe. This inoculation procedure guarantees the homogenous distribution of spores on leaf tissue at a density of 80 to 120 spores/cm2. The number of developing fungus colonies was counted 6 days after inoculation and the leaf area covered by the pathogen was estimated at 6, 7, 8 and 12 days after inoculation by image analysis using the JMicroVision software program. The infected area was considered a measure of disease severity. Statistical analyses were carried out using a random complete block design. Highly significant differences in the number of developing mildew colonies were found between plants expressing SN-1 and the non transgenic controls. The reduction of developing colony number on transgenic plants was approximately 50%. Likewise, the colonies grown on transgenic leaves showed a delay in development that rendered colonies of smaller size at the end of the experiments. An association was found between high disease resistance measured on leaf segments and high level of snakin transcripts in plant. Protection observed in those plants was similar to that observed by conventional "partial resistance" genes, which main characteristics are reduction of pustule size, smaller production of spores and longer latency period of disease development. This resistance type drives to less damage than in a susceptible host plant by retarding the development of the pathogen cycle. Likewise, the smaller amount of available fungus spores in the field along the cropping period leads to a delay in the spreading of the disease on susceptible cultivars. It is worth to mention that, to our knowledge, this is the first report on studies about the constitutive expression of the SN1 gene in wheat plants.